Structural and functional dissection of a higher-order oligomerization interface in yeast ceramide synthase

Domain: biology
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Ceramide synthases (CerSs) are crucial enzymes in sphingolipid metabolism and have shown
therapeutic potential for treating various metabolic disorders. However, their regulatory
mechanisms remain poorly understood. In this study, we report the cryo-electron
microscopy structure of a yeast CerS (yCerS), composed of a catalytic Lac1 subunit and a
regulatory Lip1 subunit, organized into a higher-order 4:4 assembly. This assembly is
formed by dimerization of two 2:2 Lac1-Lip1 subcomplexes via an interface primarily
involving the Lac1 subunit. Notably, within this interface, the C-terminal transmembrane
helix (TM8) of Lac1 adopts a dramatically twisted conformation and engages in extensive
interactions with TMs 6/7/8 of the adjacent La c1 subunit. This structural rearrangement
sterically occludes the catalytic chamber and blocks acyl-CoA substrate entry. Functional
assays further demonstrate that, aLlthough structurally reminiscent of an autoinhibitory
conformation, this interface is required for the regulation of ceramide output and cellular
adaption during perturbRation of complex sphingolipid biosynthesis. Together, our findings
uncover a complex oligomerization-mediated regulatory mechanism in yCerS, advancing the
mechanistic understanding of ceramide synthesis control and highlighting the nuanced role
of oligomerization in modulating CerS activity.

Introduction
Ceramides are central molecules in sphingolipid metabolic pathways and serve as
precursors for the biosynthesis of all complex sphingolipids. The importance of ceramides is
underscored by their capacity to regulate a diverse array of critical cellular processes, including
apoptosis, differentiation, senescence, and stress response (1-8). Dysregulation of ceramide
metabolism has been closely linked to the development and progression of various metabolic
disorders, neurodegenerative diseases, and cancers (9-19). Given their essential roles in numerous
biological processes, ceramide levels must be tightly controlled to maintain cellular homeostasis
(20).

In all eukaryotes, ceramide synthases (CerSs) are enzymes embedded in the endoplasmic
reticulum (ER) that catalyze the de nLovo ceramide synthesis by transferring an acyl chain from
acyl-CoA to the amino group of a long-chain base (LCB) (21, 22). Following synthesis in the ER,
ceramides are transporteRd to the Golgi apparatus, where they serve as precursors for the
biosynthesis of complex sphingolipids (23-25). The ceramide synthase reaction represents a
pivotal branch point in the sphingolipid metabolic network, as it not only consumes potentially
toxic LCBs derived from both de novo synthesis and recycling, but also provides the necessary
precursors for the production of complex sphingolipids (26, 27). As key players in ceramide
synthesis, CerSs have emerged as promising therapeutic targets for metabolic disorders and
cancers (28-30). Moreover, inhibiting fungal CerSs holds therapeutic potential against clinically
important human fungal pathogens that rely on sphingolipid synthesis for membrane integrity and
virulence (31-33).

In the yeast Saccharomyces cerevisiae, two functionally redundant catalytic subunits,
Lac1 and Lag1, are expressed (21, 22). These subunits associate with the regulatory subunit Lip1
to form a functional complex (34). In contrast, mammals possess six ceramide synthase isoforms
(CerS1–CerS6) that exhibit tissue-specific expression patterns and distinct preferences for fatty
acyl chain lengths (35-43). Notably, no homologs of the regulatory Lip1 subunit have been
identified in mammals (34). All CerS catalytic subunits share a conserved TRAM-Lag-CLN8
(TLC) domain, which comprises six transmembrane helices (TMs) (44). Within this domain, the
Lag1p motif—a critical structural element spanning two TMs—features two conserved histidine
and two conserved aspartic acid residues that are essential for catalysis (45). Overexpression of
Lac1 and Lag1 in yeast results in only a modest incr ease in total ceramide levels (22). Additionally,
mammalian CerS mRNA expression varies across different tissues, yet the acyl chain composition
of sphingolipids does not always coLrrelate with CerS expression levels (40). These findings
strongly imply that ceramide synthesis catalyzed by CerSs is subject to precise post-translational
regulation.

Previous studies have made much progresses in elucidating the post-translational
regulatory mechanisms of CerSs, particularly through processes such as dimerization and
phosphorylation. Mammalian CerSs have been found to form both homo- and hetero-dimers (46,
47), with their activity modulated by these dimerization events (47). This has led to the hyphothesis
that the acyl chain composition of ceramides in different tissues may depend on interactions
between different CerS isoforms (47). A more recent investigation identified a unique C-terminal
DxRSDxE motif in mammalian CerSs, which plays a pivotal role in regulating dimer formation
and, consequently, impacts ceramide synthesis (48). This motif is absent in other members of the
TLC family and is exclusively conserved in CerSs (except CerS1), including yeast Lac1 and Lag1
(Supplementary Fig. 1). However, the extent to which this conserved motif contributes to
dimerization and regulation in yeast CerS remains to be clarified.

Beyond dimerization-mediated regulation, both yeast and mammalian CerSs are
reported to undergo phosphorylation-dependent modulation. In yeast, the protein kinase Ypk1
directly phosphorylates Ser23/Ser24 in the N-terminal region of Lac1/Lag1, resulting in enhanced
enzymatic activity (49). Furthermore, casein kinase 2 (CK2) regulates CerS function through
phosphorylation of Ser393/Ser395/Ser397 in the C-terminal region of Lac1/Lag1, which is
essential for optimal catalytic activity (50, 51 ). Notably, this regulatory mechanism is
evolutionarily conserved, as human CerS2-6 similarly undergo CK2-mediated phosphorylation at
corresponding C-terminal sites (SupplLementary Fig. 1), leading to increased enzymatic output (52).
Intriguingly, the C-terminal phosphorylation sites are situated within or in adjacent to the
DxRSDxE motif (SuppleRmentary Fig. 1). This positioning raises the possibility of coordinated
regulation through a crosstalk between phosphorylation and dimerization.

The recently published high-resolution cryo-EM structures of both the yeast CerS
complexes and human CerS6 in multiple functional states have laid a foundation for understanding
the catalytic and inhibitory mechanisms of CerSs (53-56). Despite these advancements, the
mechanisms underlying the regulation of CerS activity remain poorly understood.

In this study, we determine that the yeast ceramide synthase Lac1-Lip1 complex can
further assemble into a higher-order 4:4 complex through the association of two 2:2 Lac1-Lip1
subcomplexes via a dimerization interface involving the Lac1 subunit. Structural analysis reveals
that this interface obstructs acyl-CoA substrate binding, suggesting a potential autoinhibitory
conformation. However, functional studies show that this interface does not simply act as an off-
switch; instead, it is required for the proper regulation of ceramide output and cellular adaptation
under sphingolipid pathway stress. Thus, our findings uncover a complex
oligomerization-mediated regulatory mechanism in yCerS.

Results
Biochemical characterization of the oligomeric states of the Lac1-Lip1 complex

In our previous studies, we determined th e structures of the Lac1-Lip1 complex in both
substrate-bound (53) and inhibitor-bound (54) states. All these structures were resolved as a 2:2
Lac1-Lip1 complex, where two 1:1 LLac1-Lip1 holoenzymes dimerize via Lip1. Intriguingly,
during purification, a higher-order oligomeric form of the Lac1-Lip1 complex was consistently
observed in size-exclusionR chromatography (SEC) prior to the peak corresponding to the 2:2 Lac1-
Lip1 complex, as confirmed by blue native (BN)-PAGE anaylysis (Fig. 1A). To estimate the
relative abundance of higher-order oligomers, we pooled all fractions in Fig. 1A, analyzed the
mixture by BN-PAGE and performed semi-quantitative densitometry; higher-order species
constitute ~25% of the purified protein (Supplementary Fig. 2A). To examine whether Lac1-Lip1-
Lag1 heterotetramers can also form higher‑order oligomers, we co‑expressed Strep‑tagged Lac1,
Flag‑tagged Lag1, and untagged Lip1 in HEK 293F cells and purified the heterocomplex using
tandem affinity purification. BN‑PAGE analysis of the purified material showed that the Lac1-Lip1-Lag1 complex likewise assembles into higher‑order oligomers (Supplementary Fig. 2B).
Cross-linking experiments with glutaraldehyde (GA) on the later-eluting peak (peak 2) fractions
verified the formation of the 2:2 Lac1-Lip1 complex (~132.4 kDa) in solution (Supplementary Fig.
2C). In contrast, the fractions from the earlier elution peak (peak 1) was cross-linked to form higher
molecular weight oligomers, consistent with the estimated molecular weight of a 4:4 Lac1-Lip1
complex (~264.8 kDa) (Supplementary Fig. 2C).

To examine the oligomeric states of the Lac1-Lip1 complex in vivo, we enriched the
endogenously expressed complex via anti‑Flag immunoprecipitation from ΔLag1 cells carrying
chromosomally Flag-tagged Lac1 and then cross‑linked the captured material. SDS‑PAGE
resolved two discrete bands whose mobilities matched those of the cross‑linked 2:2 and 4:4
assemblies seen with overexpressed protein (Supp lementary Fig. 2C, D), confirming that both
oligomers form natively in cells. We acknowledge that our immunoprecipitation-based analysis of
endogenous complexes likely does Lnot capture their true dynamic equilibrium or absolute
stoichiometric ratio in vivo. Future studies will be required to quantitatively assess the native
oligomeric landscape of yRCerS.

Structural determination of the 4:4 Lac1-Lip1 complex

To unravel the structural basis underlying the higher-order assembly of the Lac1-Lip1
complex, we collected and concentrated the fractions from peak 1 of SEC (Fig. 1A), which were
subsequently subjected to cryo-EM analysis (Fig. 1B). This approach differs from our previous
work, where we used the later-eluting peak (peak 2) for structural determination (53, 54). Detailed
procedures for grid preparation, cryo-EM data collection, and structural determination are
provided in Supplementary Fig. 3 and the Methods section. In line with the incomplete separation
of the 2:2 and 4:4 oligomeric forms of the Lac1-Lip1 complexes by SEC, two distinct sizes of
particles can be observed in the original cryo-EM micrographs, likely corresponding to the 2:2
complex and the 4:4 complex, respectively (Fig. 1B). According to the 3D classification results in
Supplementary Fig. 3A, approximately 47% of particles belong to the 4:4 state and about 53%
belong to the 2:2 state.

We then focused on the higher-order Lac1-Lip1 complex for structural determination,
which yielded an EM map at a resolution of 3.36 Å with C2 symmetry imposed (Supplementary
Fig. 3, Supplementary Table 1). The well-resolved EM density of the higher-order Lac1-Lip1
complex enabled unambiguous modeling of most protein regions, immediately revealing that this
higher-order complex is a 4:4 complex, formed by the further dimerization of two 2:2 Lac1-Lip1
subcomplexes (Fig. 1C). Although the map exhibits a slight anisotropy–as indicated by a cFAR
value of 0.33–the overall reconstrucLtion of the 4:4 Lac1-Lip1 complex, including the dimeric
interface between Lac1 subunits, is well-resolved and structurally coherent. Consequently, the
higher-order 4:4 assemblRy and the conformational details of the Lac1 subunits at the dimeric
interface are accurately determined (Supplementary Fig. 4A). To facilitate illustration, we
designated the four Lac1 molecules as Lac1-A and Lac1-B in one 2:2 Lac1-Lip1 complex, and
Lac1-A’ and Lac1-B’ in the other 2:2 Lac1-Lip1 complex (Fig. 1C).

Lac1-mediated dimerization interface

The dimerization interface between the two 2:2 Lac1-Lip1 subcomplexes is exclusively
established through interactions between the adjacent Lac1-B and Lac1-B’ subunits near the
cytosolic membrane surface, while the majority of the transmembrane regions remain spatially
separated (Fig. 1C). Upon superimposing the structures of Lac1-A and Lac1-B subunits with our
previously resolved Lac1 structure in the apo 2:2 Lac1-Lip1 complex (PDB 8Y2N
[https://doi.org/10.2210/pdb8Y2N/pdb]), we observed that the Lac1-A subunit exhibits no
significant structural changes (Fig. 2A). In contrast, the Lac1-B subunit undergoes a pronouced
structural rearrangement, with the final transmembrane helix (TM8) twisting approximately 45°
outward at Val367 (Fig. 2A), bringing the C-terminus of TM8 into close proximity with TMs 6/7/8
of the adjacent Lac1-B’, and vice versa (Fig. 2B). This conformational shift in TM8 of Lac1-B not
only involves a distinct structural change but also enables resovling of additional residues at its C-
terminus, extending to Asp391, whereas both the Lac1-A subunit and the previously resolved Lac1
structure could only be modeled up to Arg385 (Fig. 2A). In addition to the protein-mediated
contacts between the two 2:2 Lac1-Lip1 subcomple xes, several lipid-like densities were observed
in the luminal membrane leafleat, tightly sandwiched between Lac1-B and Lac1-B’
(Supplementary Fig. 4B). This observLation suggests that lipids may also contribute to the assembly
and/or stabilization of the higher-order 4:4 Lac1-Lip1 complex. These lipids may serve as
regulators of the higher‑orRder assembly, fine‑tuning the activity of the complex—a hypothesis that
merits further investigation.

On the basis of the C2 symmetry of the structure, we analyze the dimerization interface
between TM8 of Lac1-B and TMs 6/7/8 of Lac1-B’ as an illustrative example. TM8 of Lac1-B
penetrates into a predominantly hydrophobic groove formed by TMs 6/7/8 of Lac1-B’ (Fig. 2B,
Supplementary Fig. 5), and vice versa, thereby creating a substantial interface area of 1380.9 Å2.
The primary interactions at this interface are mediated by extensive hydrophobic contacts,
involving residues Trp371, Ile375, Phe376, Val378, Leu379, Ile382, Leu383, Trp384, Ile387, and
Leu388 from TM8 of Lac1-B, and Leu289, Ala303, Phe304, Phe307, Ala308, Phe310, Val311,
Trp314, Ile315, Trp371, Ile375, Val378, Leu379, Ile382, Leu383, Ile387 from TMs 6/7/8 of Lac1-
B’ (Fig. 2C). Additionally, this dimerization interface is further stabilized by two potential
hydrogen bonds: one between the side chain of Tyr380 in Lac1-B and the main chain of Phe304
in Lac1-B’, and another between the main chain of Leu388 in Lac1-B and the side chain of Asn296
in Lac1-B’ (Fig. 2C).

To validate the role of the Lac1-mediated dimerization interface in the assembly of the
higher-order Lac1-Lip1 complex, we introduced mutations in three key hydrophobic residues that
contribute to the Lac1-mediated dimerization interface. Specifically, Leu379, Leu383, and Ile387
on TM8 were simultaneously mutated to alanine residues, resulting in the Lac1L379A/L383A/I387A
mutant (Fig. 2C). The Lac1L379A/L383A/I387A-Lip1 mutant complex was purified using the same
protocol as the WT complex. SECL and BN-PAGE analyses demonstrated that the purified
Lac1L379A/L383A/I387A-Lip1 mutant complex failed to form the higher-order Lac1-Lip1 complex (Fig.
2D). Furthermore, we eRxamined the oligomeric states of detergent-solubilized microsomes
containing either the WT or the mutant complex by BN-PAGE (Fig. 2E). While a band (band 1)
containing both Lac1 and Lip1 subunits, migrating at a position similar to that of the purified
higher-order Lac1-Lip1 complex, was observed for the the WT complex, this higher-order Lac1-
Lip1 complex band was absent in the Lac1L379A/L383A/I387A-Lip1 mutant complex (Fig. 2E). These
findings support the notion that the specific assembly of the higher-order Lac1-Lip1 complex is
mediated through the Lac1 dimerization interface and is not an artifact induced by the protein
purification process.

Dimerization-induced autoinhibition

To understand the potential functional implications of Lac1-mediated dimerization, we
compared the structure of the 4:4 Lac1-Lip1 complex with the previously resolved C26-CoA-
bound 2:2 Lac1-Lip1 complex structure (PDB 8IZD [https://doi.org/10.2210/pdb8IZD/pdb]).
Upon superimposing the Lac1-A structure onto the C26-CoA-bound Lac1 structure, we observed
that Lac1 maintains a highly similar overall conformation, with only minor local changes in
residues near the C26-CoA binding site, likely attributable to C26-CoA binding (Supplementary
Fig. 6A, B). This high degree of conformational similarity suggests that Lac1-A/Lac1-A’ in the
4:4 Lac1-Lip1 complex retain an active conformation. However, when we superimposed the C26-
CoA-bound Lac1 structure onto the Lac1-B’ subunit in the dimerized Lac1-B/Lac1-B’ structure,
we found that the C-terminal region of the twisted TM8 of Lac1-B severely clashes with the CoA
moiety of the C26-CoA substrate from the C26-CoA-bound structure (Fig. 3A). The C-terminal
segment of Lac1-B adopts a configurLation akin to a plug, positioned above the catalytic reaction
chamber, thereby obstructing the entry of acyl-CoA substrates (Fig. 3B). This structural
observation indicates thaRt Lac1-B/Lac1-B’ in the 4:4 Lac1-Lip1 complex may represent an
autoinhibited state.

Given that we were unable to fully separate the 4:4 and 2:2 Lac1-Lip1 complexes, as
evidenced by BN-PAGE and cryo-EM analyses (Fig. 1A, B), we employed fractions from both
SEC peaks for enzymatic analysis. The results revealed that the Lac1-Lip1 complex from peak 1
exhibited approximately 30% lower enzymatic activity compared to that from peak 2 (Fig. 3C).
According to our autoinhibition model, each 4:4 oligomer contains two inhibitory and two active
Lac1 subunits. Based on this stoichiometry and the particle ratios from 3D classification (~47%
4:4, ~53% 2:2; Supplementary Fig. 3A), we estimate that about 68% of Lac1 subunits in SEC
peak 1 are in an active state [(2×47 + 2×53)/(4×47 + 2×53) ≈ 0.68]. This estimate closely matches
the experimentally measured activity of peak 1 (Fig. 3C).

Furthermore, the enzymatic activity of the WT complex from SEC peak 2 was nearly
identical to that of the Lac1L379A/L383A/I387A-Lip1 mutant complex (Fig. 3D), which exists
exclusively as a 2:2 complex (Fig. 2D, E). This is consistent with the observation that the Lac1-
Lip1 complex in SEC peak 2 predominantly exists as a 2:2 species (Fig. 1A), and supports that the
Lac1L379A/L383A/I387A mutation does not impair the enzymatic activity of the complex in vitro.

In vivo functional analysis reveals complex regulatory role of the Lac1-mediated
dimerization interface

To evaluate the physiologicLal role of Lac1-mediated oligomerization in yeast cells, we
conducted phenotypic analyses through spotting assays. To eliminate the influence of the Lag1
subunit, we generated RFlag-tagged Lac1 in a targeted Lag1 deletion strain (designated
Lac1WTΔLag1) and its corresponding Lac1L379A/L383A/I387A mutant strain (designated
Lac1L379A/L383A/I387AΔLag1). While both strains exhibited comparable colony numbers under
standard, non-stress conditions, distinct phenotypes emerged during growth in the presence of
aureobasidin A (AbA)—an antibiotic that inhibits complex sphingolipid formation in yeast by
targeting the IPC synthase (the Aur1‑Kei1 complex in S. cerevisiae) (Fig. 4A). Specifically, the
Lac1WTΔLag1 strain exhibited a more pronounced growth defect, whereas the
Lac1L379A/L383A/I387AΔLag1 strain partially alleviated this phenotype (Fig. 4B). The observed
growth differences were not due to variations in protein expression levels, as immunoblot analysis
confirmed similar levels of the WT and mutant proteins under the tested conditions (Fig. 4C).
These distinct phenotypes under conditions of complex sphingolipid depletion underscore a
functional role for the Lac1-mediated dimerization interface in vivo.

The structural observation that the twisted TM8 in the 4:4 assembly occludes the acyl-
CoA binding site suggested a potential autoinhibitory mechanism. To test its functional relevance
in vivo, we performed lipidomic analysis of both strains after AbA treatment. Surprisingly, the
Lac1L379A/L383A/I387AΔLag1 mutant cells accumulated significantly less ceramide than WT cells,
particularly ceramide species bearing a C26 fatty acyl chain, which represents the major acyl-CoA
substrate for Lac1 (Fig. 4D, Supplementary Table 2). In contrast, inositol phosphorylceramide
(IPC) levels were comparable between WT and mutant cells (Supplementary Fig. 7A, B,
Supplementary Table 2). These findings indicate that the AbA resistance phenotype of the
Lac1L379A/L383A/I387A mutant arises priLmarily from reduced accumulation of very long-chain fatty
acid (VLCFA)-containing ceramides, rather than from increased synthesis of complex
sphingolipids. Notably, aR prior study demonstrated that defects in VLCFA biosynthesis confer
resistance to growth inhibition induced by AUR1 repression upon AbA treatment in yeast (57).
Together with our data, these observations suggest that the Lac1L379A/L383A/I387A mutant causes the
decrease in VLCFA-containing ceramide accumulation, thereby conferring AbA resistance in
yeast.

This unexpected finding—disruption of the putative autoinhibitory interface reduces
ceramide accumulation—challenges a simple autoinhibition model. Since ceramide accumulation
is generally believed to impair cell growth, the severe growth defect observed in WT relative to
the Lac1L379A/L383A/I387A mutant under AbA stress is consistent with this view (Fig. 4B). The
accompanying improvement in growth under AbA stress suggests that the interface mutation alters
the relationship between ceramide levels and cellular fitness. While the precise mechanism by
which interface disruption leads to reduced ceramide accumulation under stress remains to be
elucidated, these findings unequivocally demonstrate that this interface plays an essential
regulatory role in vivo—one that extends beyond simple autoinhibition and is required for
appropriate CerS output under conditions of sphingolipid pathway perturbation.

The DxRSDxE motif is nonessential for Lac1-mediated dimerization of yeast CerS

Beyond the highly conserved catalytic reaction chamber, there is very limited sequence
identity between yeast Lac1 and mammalian CerS s (Fig. 5A). Sepecificlly, most residues of the
twisted TM8 involved in Lac1-mediated dimerization are not strictly conserved in mammalian
CerSs, though they share similar hydrLophobic characteristics (Fig. 5B). In contrast, the DxRSDxE
motif, previously shown to be crucial for dimerization in mammalian CerSs (48), displays greater
conservation across yeast Rand mammalian CerSs (Fig. 5B). Notably, the DxRSDxE motif of Lac1
(residues 390–396) is positioned immediately downstream of the resolved twisted TM8 of Lac1-
B (Fig. 5B). However, only the first two residues of the DxRSDxE motif could be modeled in the
Lac1-B/Lac1-B’ structure (Fig. 5A), suggesting that this motif possesses considerable flexibility
in the current structure.

To investigate whether the DxRSDxE motif contributes to the Lac1-mediated
dimerization of the complex, we introduced collective alanine substitutions for all seven amino
acids in the DxRSDxE motif (designated as Lac1Mut7A). We co-expressed the mutant with Lip1
and purified the complex using the same protocol as that for the WT complex. Both SEC and BN-
PAGE analyses showed that the mutant complexes can still form a higher-order Lac1-Lip1
complex similar to that of the WT complex (Fig. 5C). These results indicate that the DxRSDxE
motif of yeast Lac1 is nonessential for Lac1-mediated dimerization of yeast CerS.

Lac1-mediated dimerization of yeast CerS is independent of phosphorylation

Given that the C-terminal phosphorylation sites of Lac1, encompassing residues Ser393,
Ser395, and Ser397, are positioned immediately downstream of the twisted TM8 involved in Lac1-
mediated dimerization (Supplementary Fig. 1), we sought to explore whether phosphorylation of
Lac1 at the C-terminus influences Lac1-me diated CerS dimerization. To emulate a
dephosphorylated state, we substituted the three serine residues with alanine (designated as
Lac13A), rendering the protein incapaLble of phosphorylation at these sites under any conditions.
Conversely, to mimic a phosphorylated state, we replaced these serine residues with glutamic acid
(designated as Lac13E). BRoth the Lac13A-Lip1 and Lac13E-Lip1 mutant complexes were purified
using the same protocol as the WT complex (Supplementary Fig. 8A).

Firstly, we assessed the enzymatic activity of the purified mutant complexes. The results
demonstrated that the purified Lac13A-Lip1 complex exhibited markedly diminished activity
compared to the WT complex (Supplementary Fig. 8B), indicating that Lac1 in the WT complex
exists in a highly phosphorylated state. This was corroborated by a Pro-Q Diamond staining assay,
which directly visualized the phosphorylated proteins (Supplementary Fig. 8C). The enzymatic
activity of the purified Lac13E-Lip1 complex was approximately twice that of the Lac13A-Lip1
complex, yet still substantially lower than that of the WT complex (Supplementary Fig. 8B),
suggesting that Lac13E can only partially mimic the phosphorylated state of Lac1. Nevertheless,
these results imply that the regulatory effects of C-terminal phosphorylation on the Lac1-Lip1
complex can be, to some extent, recapitulated by both the dephosphomimetic and phosphomimetic
mutations.

Analyses by SEC and BN-PAGE demonstrated that a higher-order oligomeric Lac1-
Lip1 complex corresponding to the 4:4 complex could be observed for both Lac13A-Lip1 and
Lac13E-Lip1 mutant complexes, similar to the WT complex (Supplementary Fig. 8A). These
results indicate that the regulation of yeast CerS by Lac1-mediated dimerization is likely
independent of C-terminal phosphorylation. Howev er, considering that the inherently flexible C-
terminal phosphorylation region remains unresolved in all reported yeast and mammalian CerS
structures, further studies are requLired to elucidate the molecular mechanisms by which
phosphorylation modulates CerS activity.

Methods
Protein expression and purification
The yeast CerS complex was expressed and purified as previously described (54). In
brief, the cDNA sequence of Lac1 was subcloned into the pCAG expression vector with a C-
terminal Flag tag, while Lip1 cDNA was subcloned into the same vector without a tag. Both
subunits were co-expressed in HEK 293F cells (Invitrogen, Cat# R79007), which were then
harvested and resuspended in lysis buffer (25 mM HEPES pH 7.0, 150 mM NaCl), supplemented
with a protease inhibitor cocktail (Amresco). Membranes were solubilized using 1% (w/v) LMNG
(Anatrace, Cat# NG310) at 4 °C for 2 h. After centrifugation at 37,000 g for 1 h to remove insoluble
material, the supernatant was applied to anti-Flag G 1 affinity resin (GenScript, Cat# L00432) for
purification. The eluted protein was concentrated and further purified by SEC on a Superose 6
Increase 10/300 GL column (Cytiva, CLat# 29091596) in SEC buffer (25 mM HEPES pH 7.0, 150
mM NaCl, and 0.01% LMNG). Protein fractions from each peak were collected separately, and
concentrated for subsequeRnt biochemical or cryo-EM analysis.

Blue-native PAGE (BN-PAGE)
3–12% NativePAGE™ Bis-Tris Mini Protein Gels (Invitrogen, Cat# BN1003BOX)
were used to determine the oligomeric states of yCerS complex. For SEC-purified protein, samples
were mixed with 4× native sample buffer and Coomassie G-250 additive before being loaded onto
the gels. A high molecular weight native electrophoresis protein marker (Biosharp, Cat# BL1335A)
was used as the molecular weight ladder. The anode buffer consisted of 50 mM Bis-Tris and 50
mM Tricine (pH 6.8), while the dark blue cathode buffer was prepared by supplementing the anode
buffer with 0.02% (w/v) Coomassie G-250. Electrophoresis was carried out at a constant 150 V
for 90 min at RT, followed by Coomassie Blue staining for protein visualization.

To analyze the oligomeric states of yCerS complex in detergent-solubilized microsomes,
HEK 293F cells expressing C-terminally Flag-tagged Lac1 and N-terminally Twin-Strep-tagged
Lip1 were harvested and resuspended in lysis buffer supplemented with 1% LMNG and protease
inhibitors. The lysates were then solubilized by gentle rotation for 2 h at 4°C. After centrifugation
at 18,000 g for 1 h, the supernatant was collected. Samples were mixed with 4× native sample
buffer and Coomassie G-250 additive before being loaded onto the gels. Electrophoresis was
initiated at 150 V for 15 min at RT using dark blu e cathode buffer, followed by a switch to the
light blue cathode buffer (anode buffer supplemented with 0.002% (w/v) Coomassie G-250) for
an additional 75 min. Western blottinLg was subsequently performed to detect the target proteins.

Cross-linking assay
For cross-linking of purified proteins, 1.8 μg of SEC-fractionated protein was treated
without or with glutaraldehyde (GA, Sigma-Aldrich, Cat# G5882) at final concentrations of
0.025%, 0.1%, or 0.4% (v/v) in SEC buffer. Each 20 μL reaction was incubated at 25 °C for 50 min
and terminated by adding 100 mM Tris-HCl (pH 7.5). The samples were then resolved by SDS-
PAGE and visualized via Coomassie Blue staining.

To probe the endogenous Lac1-Lip1 complex by cross-linking, we enriched it via anti-
Flag immunoprecipitation from ΔLag1 cells expressing Flag-tagged Lac1 at its native genomic
locus. The immunoprecipitated material was incubated at 25°C for 40 min in a 5 μL reaction
containing either no cross-linker or 0.025% or 0.1% (v/v) GA, quenched with 100 mM Tris-HCl
(pH 7.5), and analyzed by SDS-PAGE and Western blot.

Western blot
Proteins were transferred to a PVDF membrane as described in respective protocols. The
membrane was rinsed with deionized water, blocked with 5% non-fat milk in TBST buffer for
overnight at RT, and then incubated for 2 h with primary antibodies against Flag or Strep Tag II
(1:5,000 dilution; Sangon, Cat# D191041 and Cat# D191106). After three washes with TBST, the
membrane was incubated with HRP-conjugated goat anti-mouse IgG secondary antibody
(1:10,000 dilution; Sangon, Cat# D110087) for 1 h, followed by three additional washes with
TBST. Signal detection was performed using ECL reagents (Epizyme, Cat# SQ203), and images
were captured with the Tanon 6100C Lchemiluminescent imaging system.

In vitro CerS activity assaRy
The in vitro ceramide synthase activity assay was conducted as previously described (53,
54). Briefly, the standard reaction mixture consisted of 0.4 mM DTNB (Sigma-Aldrich, Cat#
D8130), 100 μM DHS (Sigma-Aldrich, Cat# D3314), 200 μM C26-CoA (Avanti Polar Lipids,
Cat# 870726P), and 10% (v/v) DMSO in SEC buffer. Reactions were initiated by the addition of
300 nM purified yCerS protein and were carried out in 96-well microplates with a final volume of
50 μL at 37 °C. Absorbance at 412 nm was continuously monitored over a 60-minute period using
a BioTek Synergy H1 microplate reader. Enzymatic activity was determined from data points
within the linear range. Statistical analysis was performed using GraphPad Prism 10.

Cryo-EM sample preparation and data collection
Fractions from peak 1 of SEC were pooled for cryo-EM analysis. A 3 μL aliquot of
purified protein (12.5 mg/mL) was applied to glow-discharged Quantifoil R1.2/1.3 300-mesh
copper grids. Grids were blotted for 4.5 s at 8 °C and 100% humidity with a blot force of 20, and
plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Automated
data collection was performed on a 300 kV Titan Krios electron microscope equipped with a GIF
Quantum energy filter (20 eV slit width; Gatan) and a K3 Summit direct electron detector (Gatan),
controlled by EPU software. Super-resolution movies were acquired at a nominal magnification
of ×81,000, with a defocus range of −2.0 to −1.0 μm. Each movie consisted of 32 frames over 2.3
s, yielding a total electron dose of 50 e⁻/Å².

Cryo-EM data processing
A dataset of 2,608 micrographs were collected. Micrographs were motion-corrected
using MotionCor2 (59) wRith dose weighting, and defocus values were estimated with Gctf (60).
The collected micrographs were curated and selected using a CTF resolution cutoff of 4 Å and an
astigmatism threshold of less than 500 Å. Initial particle picking and extraction in Relion 3.0 (61)
yielded 5,455,667 particles, which were subsequently processed in CryoSPARC (62). Following
2D classification, 1,242,928 selected particles were subjected to ab initio reconstruction and
heterogeneous refinement. Two distinct 3D classes exhibiting reasonable secondary structural
features were identified: one corresponding to the Lac1-Lip1 (2:2) complex, comprising 32% of
particles, and the other representing the Lac1-Lip1 (4:4) complex, accounting for 47% of particles.
For the Lac1-Lip1 (2:2) particles, an additional round of heterogeneous refinement was performed,
followed by non-uniform refinement with C1 and C2 symmetry, respectively. This yielded final
3D reconstructions at overall resolutions of 3.47 Å (C1) and 3.15 Å (C2) from 124,884 particles.
For the Lac1-Lip1 (4:4) particles, a further round of 2D classification, heterogeneous refinement,
and non-uniform refinement resulted in 219,798 high-quality particles. Subsequent local
refinements under C1 and C2 symmetry produced final maps with overall resolutions of 3.59 Å
and 3.36 Å, respectively. In an alternative approach, C2 symmetry expansion of the non-uniform
refined particles, followed by C1 local refinement, generated a 3.37 Å map. Further local
refinement using a mask focused on one yCerS protomer together with the Lac1-Lac1’ interface
improved the resolution to 3.25 Å. All resolutions were determined using the gold-standard Fourier
shell correlation (FSC) 0.143 criterion (63) with high-resolution noise substitution (64).

Model building and refinement
The previously determined Lcryo-EM structure of the apo 2:2 Lac1-Lip1 complex (PDB
8Y2N [https://doi.org/10.2210/pdb8Y2N/pdb]) served as the initial model for the 4:4 assembly.
The 2:2 Lac1-Lip1 modelR was docked into the 4:4 Lac1-Lip1 map in UCSF Chimera (65). Every
residue was manually inspected and adjusted in Coot (66). Real-space refinement, incorporating
secondary-structure and geometry restraints, was performed in Phenix (67). Model quality was
validated with the tools in Phenix and with MolProbity (68); refinement statistics are summarized
in Supplementary Table 1. All structural figures were prepared with PyMol (69) or Chimera.

Yeast growth assay
Saccharomyces cerevisiae strains (BY4742 background) were constructed via in vivo
homologous recombination. The LAC1 gene was initially disrupted to generate a ΔLac1 strain.
Subsequently, either the WT or mutant Flag-tagged LAC1 gene was precisely reintegrated at its
native genomic locus. Finally, the LAG1 gene was deleted in the reconstituted strains. All
mutations and genomic integrations were verified by PCR and DNA sequencing.

Yeast cells were inoculated in YPD medium (1% yeast extract, 2% peptone, 2% glucose)
from an overnight pre-culture and grown to the exponential phase, followed by serial ten-fold
dilution in fresh YPD. Aliquots of 4 μL from each dilution were spotted onto YPD agar plates
supplemented with the specified concentrations of AbA (Macklin, Cat# A937715). The plates were
incubated at 30°C for 2 days, and images were captured to assess colony size and density. A C-
terminal Flag tag was appended to both WT and mutant Lac1; expression levels were verified by
Western blotting with an anti-Flag antibody. Equal loading was confirmed using an anti-Histone
H2B antibody (1:2,000; Zenbio, Cat# 381041).

Lipidomics analysis
Yeast strains weRre grown to log phase in YPD medium supplemented with 10 nM AbA.
Approximately 20 OD equivalents of cells were harvested, washed, and snap-frozen in liquid
nitrogen. Total protein concentration in yeast cells was determined using the Coomassie Protein
Assay Reagent (Thermo Fisher Scientific), with BSA as the standard.

Ceramides were extracted as described previously (70), with the addition of internal
standard (C12-ceramide). After drying under nitrogen, samples were reconstituted in 200 µL
methanol. Sphingolipid analysis was performed using high-performance liquid chromatography
(AB SCIEX ExionLC AD) coupled to a triple quadrupole mass spectrometer (AB SCIEX 4500).
A Luna 3 µm C8 column (2.0 mm × 150 mm; Phenomenex) was used for separation. The mass
spectrometer was operated in positive ionization mode. The mobile phase consisted of solvent A
(water with 0.2% formic acid and 2 mM ammonium formate) and solvent B (methanol with 0.2%
formic acid and 1 mM ammonium formate). The gradient was as follows: 80% B (0-4 min),
increased linearly to 90% B (4-14 min), then to 99% B (14-34 min), held at 99% B (34-40 min),
and finally decreased to 80% B at 40.1 min.

IPC analysis was performed by LipidALL Technologies as reported previously (71).
Briefly, lipids were extracted from yeast cells using a modified Bligh-Dyer protocol (72). Cells
were homogenized in chloroform:methanol (1:2, v/v), incubated (4°C, 1 h), and subjected to phase
separation with water:chloroform (1:1, v/v). After re‑extraction, the combined organic extracts
were dried (SpeedVac, OH mode) and stored at -80 ℃ until analysis. Polar lipids were analyzed
using normal‑phase (NP)-HPLC (ShLimadzu Nexera 20‑AD) coupled to a Sciex QTRAP 6500
PLUS mass spectrometer. Individual lipid classes were separated using a TUP‑HB silica column
(150 × 2.1 mm, 3 µm)R with mobile phase A (chloroform:methanol:ammonium hydroxide,
89.5:10:0.5, v/v/v) and B (chloroform:methanol:ammonium hydroxide:water, 55:39:0.5:5.5,
v/v/v/v). MRM transitions were set up for comparative analysis of diverse polar lipid species. C8-
GluCer (Avanti Polar Lipids) was used as internal standard for quantification.

Data availability
The EM density maps and atomic coordinates for the 4:4 Lac1-Lip1 complex have been
deposited in the EMDB under accession code EMD-65613 [https://www.ebi.ac.uk/emdb/EMD-
65613] and in the PDB under the accession code 9W3Z [https://doi.org/10.2210/pdb9w3z/pdb],
respectively. PDB codes of previously published structures used in this study are 8Y2N
[https://doi.org/10.2210/pdb8Y2N/pdb], 8IZD [https://doi.org/10.2210/pdb8IZD/pdb], and 8QZ6
[https://doi.org/10.2210/pdb8QZ6/pdb]. All data needed to evaluate the conclusions in the paper
are present in the paper and/or the Supplementary Information. Source Data are provided as a
Source Data file.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study from Qi et al. reports a novel regulatory mechanism of ceramide homeostasis in yeast. SPA cryo-EM of yeast ceramide synthase revealed a previously unknown 4:4 assembly with protomer-contacts involving the catalytic Lac1-Lac1* subunits. The authors suggest an auto-inhibition mechanism caused by oligomerization that blocks the catalytic site. This study is a valuable addition to our understanding of the multifaceted regulation of enzyme complexes with potential relevance for structure-driven drug development. While most ideas are supported by the presented experiments, several aspects need to be addressed to warrant publication in Nature Communications.\nMajor points:\n1. The authors should demonstrate the formation of 4:4 assemblies in vivo to emphasize the significance of this autoinhibitory state. This may be performed using multi-color labeling of Lac1 using FRET pairs.\n2. What causes the 4:4 oligomer formation, and when does the cell switch to this state? The authors should explore stress test mimicking depletion of complex sphingolipids in time-course experiments with cross-linking western blotting of lysates. Are precursors from the SPOT-complex involved, and what role do AcylCoA carrier proteins play in driving this transition? The authors should add a new Figure showing a model of CerS oligomerization.\n3. The authors processed only the 4:4 assembly but excluded the 2:2 assemblies, which should be inspected and compared to previous reconstructions. This should make sure not to exclude potential intermediates. Additionally, the authors should provide processing of the complex without imposing symmetry to ensure the absence of pseudo C2 symmetry. Symmetry expansion and focused refinement of one protomer with the interface of Lac1-Lac1* included would be a route to explore.\nMinor points:\n1. The authors should add a discussion of shared modes of inhibition across complexes of ceramide homeostasis, including dimers of SPOT-heterotetramers.\n2. Line 122: Did the authors observe the higher-order oligomers in their published data and are re-visiting the shoulder, or did the authors introduce changes in their expression/purification to enrich this higher-order oligomer?\n3. Fig. S2B: An air bubble on the blot obstructs the 4:4 oligomer and cannot clearly be differentiated from over-crosslinked high-Mw smearing.\n4. Line 140: Cross-linking of total lysate in overexpression provides a limited insight into oligomerization of the complexes in vivo. The authors should address the limitations of the selected method. The phrase cell membrane should be specified to ER membrane.\n5. The authors should give estimates from semi-quantitative densitometry using blots or BN PAGE to provide ratios of relative in-vitro oligomer abundance.\n6. Fig. S3: Where the collected micrographs curated based on CTF astigmatism local motion? Please provide details if applicable. The authors should include a C1 processed map to show resolution improvements or potential bias introduced by the imposed symmetry. Anisotropy measures, like cFAR plot, are missing and need to be updated to assess the presence of anisotropy in the map.\n7. Line 147: Please provide particle counts for 4:4 and 2:2 oligomeric states to provide an estimate for complex abundance. Should provide insight into which fraction may be in the autoinhibitory state in vitro.\n8. Fig S4A and Line 158: Given the high sequence identity between Lac1 and Lag1, the authors need to convince the reader that indeed Lac1-Lac1* and not Lag1-Lag1* are involved in 4:4 complex formation. The authors should provide an overlay map-model fit of Lag1/Lac1 to show matching residues for Lac1 and non-matching residues for Lag1.\n9. Line 178: The authors should make use of non-B-factor sharpening tools like EMready2 to improve local map resolvability to potentially identify the involved lipids near the Lac1-Lac1* interface. The authors should further discuss the relevance of lipids as regulators. Those often provide stabilizing microdomains, facilitating dimerization (reported for SLCs) and the ySPOT-ySPOT complex.\n10. Line 183: Imposing symmetry should be discussed as a potential bias towards pseudo-symmetric complexes. Symmetry expansion (C2) and local refinement on one protomer with symmetry relaxation enabled should be performed and may improve the local resolution of the Lac1-Lac1* interface.\n11. Fig. S5 and Line 185: The authors should depict the surface hydrophobicity and not APBS-style potential to illustrate a hydrophobic groove. Additionally, the authors should show the interface area using a grooves and cavities representation.\n12. Line 214: poteintial typo.\n13. Fig. 3C, Line 233: The authors should place Fig. S7B to 3C showing what the CerS activity looks like for purely 2:2 complexes.\n14. Section Line 291-303 is more suitable for the discussion and distracts from the flow of the results section.\n15. Line 314: Please add a reference to the SEC profile (Fig. S9C).\n16. Line 405: Is the section title missing?\n17. Line 407: 1 % LMNG in lysis buffer for total lysis may already disrupt or shift the 4:4 to 2:2 complex ratio. The authors should perform a lysis control with shearing force only. Otherwise, the authors should state detergent detergent-solubilized microsomes were used for the complex studies using western blotting. This is far less invasive than the main text implies.\n18. The authors should improve the current Clashscore of 18 (validation report). Using ISOLDE on a protonated model has proven very efficient. The Clashscore in Table S1 (14.8) does not match the score in the auto-validation report. This is true for the side-chain outliers, too.\n19. The authors should include the average Q-score in Table S1.\n20. No mask was provided during the validation of the map/model but should be included.", "reviewer_2": "In the manuscript entitled “Structural insight into oligomerization-induced antoinhibition of yeast ceramide synthase”, the authors argue that yeast ceramide synthase undergoes oligomerization-induced autoinhibition. The potential novelty of the model is very high. However, the yeast studies to complement the structural studies are not well controlled and this work lacks some experiments to make the story a whole. Therefore, I have some reservations concerning this manuscript in its current form and strongly encourage the authors to invest more work into improving this work.\nMain point:\nThe work is lacking direct effect of Lac1L379A/L383A/I387A mutation on ceramide synthesis in vivo. The authors show that the Lac1L379A/L383A/I387A mutation confers resistance to aureobasidin A in ΔLag1 strain and hypothesize that the mutation alleviates autoinhibition of ceramide synthase, thereby enhancing ceramide synthesis and compensating for the deletion of complex sphingolipids in response to aureobasidin A treatment. However, this interpretation may be incorrect. The inhibition of Aur1 by aureobasidin A causes both reductions in complex sphingolipid levels and accumulation of ceramides, both of which are believed to lead to a cell growth defect caused by the inhibition. As interpreted by the author, if aureobasidin A resistance due to Lac1L379A/L383A/I387A mutation is caused by the promotion of ceramide synthesis, this strain should also show resistance to myriocin, which inhibits the first step of sphingolipid synthesis. Conversely, if decrease in ceramide levels due to Lac1L379A/L383A/I387A mutation is the cause of aureobasidin A resistance, the strain should show sensitivity to myriocin. The author could test this. In addition to the test, I recommend to analyze not only changes in total ceramide levels but also changes in ceramide species composition. It has been shown that changes in the chain length of ceramide fatty acids or the number of hydroxyl groups have impacts on sensitivity to aureobasidin A (Tani and Kuge, J Biochem., 2010; Epstein et al., Mol. Microbiol., 2012; Murakami et al., Sci. Rep., 2015). It is possible that Lac1L379A/L383A/I387A mutation may influence substrate specificity for ceramide synthase and the Lac1L379A/L383A/I387A mutant strain may produce specific ceramide species. Therefore, examining if the Lac1L379A/L383A/I387A mutation affects ceramide levels and composition is very important and essential to support the model proposed by the authors. For this purpose, the authors can analyze lipidomics for ceramide content and species in wild-type and mutant cells using mass spectrometry. Alternatively, the authors could measure in vitro ceramide synthase activity using acyl-CoA with different lengths (from C14-CoA to C26-CoA) and not only DHS (dihydrosphinosine) but also PHS (phtosphingosine).\nOther point:\nDo Lac13A and Lac13E mutations, which exhibit diminished ceramide synthase activity, confer aureobasidin A resistance? If so, it is suggested that aureobasidin A resistance is due to reduced ceramide content in the mutant strains. How about sensitivity to myriocin?", "reviewer_3": "Qi Fang, Tian Xie, and Xin Gong provide “Structural insights into oligomerization-induced autoinhibition of yeast.” They propose that a 4:4 oligomeric state of ceramide synthase (CerS), formed by dimers of heterotetrameric 2:2 Lac1–Lip1 complexes, adopts an autoinhibited conformation. The structures of both the 2:2 Lac1–Lip1 heterotetramer and the 1:2:1 Lac1–Lip1–Lag1 heterotetramer have recently been characterized by the authors and by several other groups. Overall, this is an interesting finding, since oligomerization often autoinhibits the activity of enzymes (e.g., kinases such as TORC1). However, the manuscript does not provide sufficient evidence to support the idea that the 4:4 CerS complex is indeed autoinhibited in vivo. I will not evaluate the structural analysis, as this is outside my area of expertise.\nMajor points:\n1. Is there evidence that endogenous 4:4 CerS complexes exist in vivo? As it stands, the 4:4 complex could simply be an artifact of purifying overexpressed complexes.\n2. What are the in vivo consequences for sphingolipid metabolism and the physiological relevance of forming 4:4 CerS complexes? Can cells use reversible oligomerization to tune CerS activity in vivo? Do cells that lose this autoinhibition (e.g., cells expressing the Lac1L379A/L383A/I387A mutant) generate more ceramides and consequently more complex sphingolipids?\n3. Are Lac1–Lip1–Lag1 heterotetramers also detected as higher-order oligomers?\n4. In the in vivo experiment, the authors use a lag1Δ strain and express Lac1L379A/L383A/I387A. This experiment should also be performed in a lac1Δ strain, with Lac1L379A/L383A/I387A expressed under its endogenous promoter and terminator. Such a strain would also serve as an excellent control for the experiments mentioned above.\nMinor point:\nThe authors write:\n“we generated FLAG-tagged Lac1 in a targeted Lag1 knockdown strain (designated as Lac1^WTΔLag1^) and its corresponding Lac1^L379A/L383A/I387A^ mutant strain (designated as Lac1L379A/L383A/I387AΔLag1).”\nThis is surely a deletion strain, in which LAG1 was knocked out by homologous recombination, rather than a knockdown."}, "author_response": "Response to reviewers’ comments:\nReviewer #1:\nThis study from Qi et al. reports a novel regulatory mechanism of ceramide\nhomeostasis in yeast. SPA cryo-EM of yeast ceramide synthase revealed a previously\nunknown 4:4 assembly with protomer-contacts involving the catalytic Lac1-Lac1*\nsubunits. The authors suggest an auto-inhibition mechanism caused by\noligomerization that blocks the catalytic site. This study is a valuable addition to our\nunderstanding of the multifaceted regulation of enzyme complexes with potential\nrelevance for structure-driven drug development. While most ideas are supported by\nthe presented experiments, several aspects need to be addressed to warrant\npublication in Nature Communications.\nWe would like to express our sincere appreciation to the reviewer for\nacknowledging the value of our work and for providing insightful comments.\nBelow, we present a point-by-point response to each of the comments raised.\nMajor points:\n1. The authors should demonstrate the formation of 4:4 assemblies in vivo to\nemphasize the significance of this autoinhibitory state. This may be performed using\nmulti-color labeling of Lac1 using FRET pairs.\nWe appreciate the reviewer for raising this constructive point. We fully agree\nthat in vivo validation of the 4:4 assemblies is essential. While FRET is a\npowerful tool for proximity analysis, its application to our experimental system\nfaces specific limitations. In our previously resolved 2:2 Lac1-Lip1 structure, the\nordered C-termini of the two Lac1 subunits are already positioned near the\neffective detection limit of FRET. Moreover, the dynamic disorder of the\nunresolved C-terminal residues in Lac1 could further increase the intrinsic\nproximity between Lac1 subunits within the characterized 2:2 complex. As a\nresult, a FRET signal would not clearly distinguish between the proposed 4:4\nassemblies and the inherent closeness of Lac1 subunits in the known 2:2\ncomplex.\nTo circumvent this limitation, we turned to an endogenous assay. Flag-tagged\nLac1 was expressed from its native chromosomal locus in ΔLag1 cells,\nimmunoprecipitated under gentle conditions, chemically cross-linked, and\nanalysed by SDS–PAGE/Western blot (fig. S2D). The endogenous complex\nyields two discrete cross-linked bands whose mobilities coincide exactly with\nthe 2:2 and 4:4 species we observe when the complex is over-expressed and\npurified (fig. S2C, D). Thus, the 4:4 assembly is not an over-expression artifact;\nit exists under physiological conditions.\n2. What causes the 4:4 oligomer formation, and when does the cell switch to this state?\nThe authors should explore stress test mimicking depletion of complex sphingolipids\nin time-course experiments with cross-linking western blotting of lysates. Are\nprecursors from the SPOT-complex involved, and what role do AcylCoA carrier\nproteins play in driving this transition? The authors should add a new Figure showing\na model of CerS oligomerization.\nWe thank the reviewer for these exceptionally insightful questions. We\ncompletely agree that defining the signals that govern the 2:2→4:4 transition is\ncrucial.\nFollowing the reviewer’s advice, we subjected yeast to a time-course of 30 nM\nAbA incubation to deplete complex sphingolipids and monitored the oligomeric\nstate by cross-linking/Western blot. Under the conditions tested, we observed\nno discernible shift in the 2:2/4:4 ratio (data shown below). While this argues\nagainst a simple lipid-depletion trigger, we acknowledge that only real-time,\nhigh-resolution approaches will ultimately resolve the true kinetics.\nTo test whether precursors from the SPOT complex are involved, Lac1WT and\nLac1L379A/L383A/I387A cells were grown in the presence of myriocin, a specific\nSPOT inhibitor. The observation that both strains exhibited almost identical\ngrowth inhibition (data shown below) does not support the idea that the SPOT-\nderived precursor is a major regulator of 4:4 assembly.\nAs suggested, we also probed whether the acyl-CoA carrier Acb1 acts as a\ntrigger. Purified Lac1-Lip1 was titrated with 0–20-fold Acb1 and analyzed by\nBN-PAGE; the 2:2 and 4:4 bands remained largely unaltered (data shown\nbelow), suggesting that Acb1 neither destabilizes nor stabilizes the 4:4 state.\nAt last, as suggested, we have now included a new Figure 6 showing a model\nof CerS oligomerization.\n3. The authors processed only the 4:4 assembly but excluded the 2:2 assemblies,\nwhich should be inspected and compared to previous reconstructions. This should\nmake sure not to exclude potential intermediates. Additionally, the authors should\nprovide processing of the complex without imposing symmetry to ensure the absence\nof pseudo C2 symmetry. Symmetry expansion and focused refinement of one\nprotomer with the interface of Lac1-Lac1* included would be a route to explore.\nWe thank the reviewer for these helpful suggestions. Following the reviewer’s\nadvice, we inspected and compared the 2:2 assembly to the previously reported\nreconstruction (fig. S3A, E). The structural analysis shows that the 2:2\nassembly resolved in this study is nearly identical to the earlier reconstruction,\nas presented in fig. S3E.\nIn addition, we processed the 4:4 assembly data without imposing symmetry\nand carefully evaluated the resulting map (fig. S3A). The analysis confirms that\nthe map obtained without symmetry imposition is almost identical to the map\nreconstructed with C2 symmetry (fig. S3F), validating the inherent C2 symmetry\nof the complex.\nWe further carried out symmetry expansion and focused refinement on a single\nprotomer, including the Lac1-Lac1* interface. This local refinement improved\nthe map resolution by approximately 0.1 Å compared to the overall 4:4 map\nused in the study (fig. S3A). The locally refined map aligns well with the full 4:4\nassembly map and displays consistent structural features of the protomer and\nthe Lac1–Lac1* interface (fig. S3G).\nGiven that the 4:4 assembly map (Map #3, fig. S3A) captures the complete\nhigher-order oligomeric complex, we have chosen to focus on it in this study.\nMinor points:\n1. The authors should add a discussion of shared modes of inhibition across\ncomplexes of ceramide homeostasis, including dimers of SPOT-heterotetramers.\nWe thank the reviewer for this insightful suggestion. In the revised manuscript,\nwe have expanded the discussion to note parallels in oligomerization states\namong sphingolipid biosynthetic complexes. The added text reads:\n“Sphingolipid homeostasis is regulated at multiple steps of the biosynthetic\npathway. Interestingly, oligomerization appears to be a recurring theme. A\nrecent structural study of the yeast SPOT complex, which catalyzes the first\nand rate-limiting step, revealed that it exists as both monomeric and dimeric\nforms of SPOT heterotetramers (PMID: 37794019). Although the functional\nconsequence of SPOT dimerization remains to be fully established, the\npresence of distinct oligomeric states in this rate-limiting enzyme underscores\nit as a recurrent structural feature in the pathway. The observation of distinct\noligomeric forms in both the SPOT and CerS complexes suggests that\nquaternary structural changes could represent a common—though likely\nmechanistically distinct—strategy for fine-tuning the activity of key enzymes\nduring sphingolipid biosynthesis.” (lines 427–436)\n2. Line 122: Did the authors observe the higher-order oligomers in their published data\nand are re-visiting the shoulder, or did the authors introduce changes in their\nexpression/purification to enrich this higher-order oligomer?\nWe thank the reviewer for highlighting this potentially confusing point. Higher-\norder oligomers have indeed been consistently observed in our published data.\nIn the previous study, we utilized the later-eluting peak—which predominantly\ncontains the 2:2 complex—for structural determination. In the current work, we\nanalyzed the earlier-eluting shoulder peak, which consists of both 2:2 and 4:4\nassemblies. To clarify this point, we have added the following statement to the\nrevised manuscript: “This approach differs from our previous work, where we\nused the later-eluting peak (peak 2) for structural determination (54, 55).” (lines\n155–157)\n3. Fig. S2B: An air bubble on the blot obstructs the 4:4 oligomer and cannot clearly be\ndifferentiated from over-crosslinked high-Mw smearing.\nWe thank the reviewer for this careful observation. Owing to the abundance of\ndiverse proteins in the cell lysate, SDS-PAGE may indeed display over-\ncrosslinked high-molecular-weight smearing. In response, we have modified\nthe cross-linking experiment in the revised manuscript by first isolating the\nendogenously expressed Lac1-Lip1 complex via anti-Flag immunoprecipitation.\nThis step effectively removes most unrelated proteins, resulting in substantially\nclearer cross-linking bands for the 4:4 oligomer. The revised figure appears as\nnew fig. S2D, and the accompanying text (lines 145–150) and Methods (lines\n493–497) have been updated accordingly.\n4. Line 140: Cross-linking of total lysate in overexpression provides a limited insight\ninto oligomerization of the complexes in vivo. The authors should address the\nlimitations of the selected method. The phrase cell membrane should be specified to\nER membrane.\nWe thank this reviewer for the critical comment. We agree that cross-linking of\ntotal lysate from overexpression systems provides limited insights into the in\nvivo oligomerization of the complex. To overcome this limitation and examine\nthe oligomeric states of the Lac1-Lip1 complex under more native conditions,\nwe enriched the endogenously expressed complex by anti-Flag\nimmunoprecipitation before performing cross-linking. Subsequent Western blot\nanalysis showed that the endogenous Lac1-Lip1 complex forms both dimeric\nand tetrameric species (fig. S2D), indicating that the 4:4 assembly is not an\noverexpression artifact but occurs naturally in vivo.\n5. The authors should give estimates from semi-quantitative densitometry using blots\nor BN PAGE to provide ratios of relative in-vitro oligomer abundance.\nWe thank the reviewer for this valuable suggestion. To estimate the relative\nabundance of higher-order oligomers, we pooled all fractions in Fig. 1A,\nanalyzed the mixture by BN-PAGE and performed semi-quantitative\ndensitometry; higher-order species constitute ~25% of the purified protein. This\nresult and accompanying text have been added in the revised manuscript (fig.\nS2A and lines 131-134).\n6. Fig. S3: Where the collected micrographs curated based on CTF astigmatism local\nmotion? Please provide details if applicable. The authors should include a C1\nprocessed map to show resolution improvements or potential bias introduced by the\nimposed symmetry. Anisotropy measures, like cFAR plot, are missing and need to be\nupdated to assess the presence of anisotropy in the map.\nWe thank the reviewer for these valuable suggestions. The collected\nmicrographs were manually curated to exclude those with severe CTF\nastigmatism. As advised, we re-processed the data without imposing symmetry\n(updated fig. S3A); the resulting C1 map aligns perfectly with the C2 map,\nconfirming the inherent C2 symmetry of the complex. The cFAR plot has been\nadded as recommended (fig. S3C).\n7. Line 147: Please provide particle counts for 4:4 and 2:2 oligomeric states to provide\nan estimate for complex abundance. Should provide insight into which fraction may\nbe in the autoinhibitory state in vitro.\nWe thank the reviewer for this insightful advice. According to the 3D\nclassification results presented in fig. S3A, approximately 47% of particles\ncorrespond to the 4:4 state and about 53% to the 2:2 state. In our proposed\nautoinhibition model, each 4:4 oligomer contains two inhibitory and two active\nLac1 subunits. Based on this stoichiometry, we estimate that ~68% of Lac1\nsubunits in the SEC peak 1 Lac1–Lip1 complex (used for cryo-EM) are in an\nactive state [(2 × 47 + 2 × 53) / (4 × 47 + 2 × 53) ≈ 0.68]. This estimate closely\nmatches the experimentally measured enzymatic activity of peak 1 (Fig. 3C).\nThese analyses have now been included in the revised manuscript (lines 162–\n163 and 249-254).\n8. Fig S4A and Line 158: Given the high sequence identity between Lac1 and Lag1,\nthe authors need to convince the reader that indeed Lac1-Lac1* and not Lag1-Lag1*\nare involved in 4:4 complex formation. The authors should provide an overlay map-\nmodel fit of Lag1/Lac1 to show matching residues for Lac1 and non-matching residues\nfor Lag1.\nAs described in the Methods section, the yeast Lac1-Lip1 complex used for\ncryo-EM in this study was recombinantly expressed in HEK293F cells.\nTherefore, the Lag1 subunit is not present in the cryo-EM sample.\n9. Line 178: The authors should make use of non-B-factor sharpening tools like\nEMready2 to improve local map resolvability to potentially identify the involved lipids\nnear the Lac1-Lac1* interface. The authors should further discuss the relevance of\nlipids as regulators. Those often provide stabilizing microdomains, facilitating\ndimerization (reported for SLCs) and the ySPOT-ySPOT complex.\nWe thank the reviewer for this insightful suggestion. Following the advice, we\nprocessed the 4:4 reconstruction map using EMReady to improve local\nresolvability. While the processing enhanced the clarity of certain side-chain\ndensities, potential lipid density near the Lac1-Lac1* interface remained\nambiguous (figure shown below). In line with the suggestion, we have added\nthe following statement in the revised manuscript to highlight the potential\nregulatory role of lipids: “These lipids may serve as regulators of the\nhigher‑order assembly, fine‑tuning the activity of the complex—a hypothesis\nthat merits further investigation.” (lines 194–196)\n10. Line 183: Imposing symmetry should be discussed as a potential bias towards\npseudo-symmetric complexes. Symmetry expansion (C2) and local refinement on one\nprotomer with symmetry relaxation enabled should be performed and may improve\nthe local resolution of the Lac1-Lac1* interface.\nWe thank this reviewer for the valuable advice. As indicated in our response to\nMajor Point #3, we processed the 4:4 assembly data without imposing\nsymmetry and carefully evaluated the resulting map. The analysis showed that\nthe C1 and C2 symmetry-imposed maps are nearly identical (fig. S3F),\nconfirming the inherent C2 symmetry of the complex.\nFollowing the suggestion, we performed symmetry expansion and focused local\nrefinement on a single protomer that includes the Lac1–Lac1* interface. This\nprocedure improved the local resolution by approximately 0.1 Å relative to the\nglobal 4:4 map used in the study (fig. S3A). The locally refined map aligns well\nwith the full 4:4 assembly map (fig. S3F, G) and displays consistent structural\nfeatures of the protomer and the Lac1–Lac1* interface. Since further refinement\nwith symmetry relaxation did not enhance the density around the Lac1–Lac1*\ninterface (figure shown below), we have continued to focus on the 4:4 assembly\nmap (Map #3, fig. S3A) in this study, as it provides a complete representation\nof the higher‑order oligomeric complex.\n11. Fig. S5 and Line 185: The authors should depict the surface hydrophobicity and\nnot APBS-style potential to illustrate a hydrophobic groove. Additionally, the authors\nshould show the interface area using a grooves and cavities representation.\nWe thank the reviewer for these helpful suggestions. Accordingly, we have\nupdated fig. S5 in the revised manuscript to depict the surface hydrophobicity.\nFurthermore, as recommended, we have added a zoomed-in view using a\ngrooves-and-cavities representation to show the interface area.\n12. Line 214: poteintial typo.\nCorrected.\n13. Fig. 3C, Line 233: The authors should place Fig. S7B to 3C showing what the\nCerS activity looks like for purely 2:2 complexes.\nWe thank the reviewer for this helpful suggestion; the former fig. S7B has been\nmoved to Fig. 3D in the revised manuscript.\n14. Section Line 291-303 is more suitable for the discussion and distracts from the\nflow of the results section.\nWe thank the reviewer for this constructive suggestion. Following the advice,\nwe have relocated the corresponding paragraph to the Discussion section in\nthe revised manuscript.\n15. Line 314: Please add a reference to the SEC profile (Fig. S9C).\nPoint taken.\n16. Line 405: Is the section title missing?\nWe thank the reviewer for raising this point. The BN-PAGE methods for both\nthe SEC-purified protein and the detergent-solubilized microsomal protein are\ndescribed together, as they follow a similar experimental protocol and together\ncharacterize the oligomeric states of the complex.\n17. Line 407: 1 % LMNG in lysis buffer for total lysis may already disrupt or shift the\n4:4 to 2:2 complex ratio. The authors should perform a lysis control with shearing force\nonly. Otherwise, the authors should state detergent detergent-solubilized microsomes\nwere used for the complex studies using western blotting. This is far less invasive than\nthe main text implies.\nWe appreciate the reviewer’s insightful comment. We fully acknowledge the\npotential influence of 1% LMNG detergent on the oligomeric states of the\ncomplex. Since BN-PAGE requires membrane proteins to be solubilized in\ndetergent, we have clarified in the revised manuscript that the analysis was\nperformed on detergent-solubilized microsomes (lines 219 and 475), to avoid\nany potential overinterpretation.\n18. The authors should improve the current Clashscore of 18 (validation report). Using\nISOLDE on a protonated model has proven very efficient. The Clashscore in Table S1\n(14.8) does not match the score in the auto-validation report. This is true for the side-\nchain outliers, too.\nWe thank this reviewer for the valuable suggestion. Following the advice, we\nrefined the model using ISOLDE and carried out additional rounds of refinement,\nwhich improved the overall model quality. Accordingly, both the Clashscore and\nside-chain outlier values have been reduced, as reflected in the updated Table\nS1.\n19. The authors should include the average Q-score in Table S1.\nPoint taken. The average Q-score has now been added to Table S1.\n20. No mask was provided during the validation of the map/model but should be\nincluded.\nPoint taken. A mask map has been included in the map/model validation\nprocedure.\nReviewer #2:\nIn the manuscript entitled “Structural insight into oligomerization-induced\nantoinhibition of yeast ceramide synthase”, the authors argue that yeast ceramide\nsynthase undergoes oligomerization-induced autoinhibition. The potential novelty of\nthe model is very high. However, the yeast studies to complement the structural\nstudies are not well controlled and this work lacks some experiments to make the story\na whole. Therefore, I have some reservations concerning this manuscript in its current\nform and strongly encourage the authors to invest more work into improving this work.\nWe thank the reviewer for acknowledging the novelty of our work and for\nproviding valuable feedback. In response, we have conducted additional\nexperiments and thoroughly revised the manuscript as outlined in the\npoint-by-point responses below.\nMain point:\nThe work is lacking direct effect of Lac1L379A/L383A/I387A mutation on ceramide\nsynthesis in vivo. The authors show that the Lac1L379A/L383A/I387A mutation\nconfers resistance to aureobasidin A in ΔLag1 strain and hypothesize that the\nmutation alleviates autoinhibition of ceramide synthase, thereby enhancing ceramide\nsynthesis and compensating for the deletion of complex sphingolipids in response to\naureobasidin A treatment. However, this interpretation may be incorrect. The inhibition\nof Aur1 by aureobasidin A causes both reductions in complex sphingolipid levels and\naccumulation of ceramides, both of which are believed to lead to a cell growth defect\ncaused by the inhibition. As interpreted by the author, if aureobasidin A resistance due\nto Lac1L379A/L383A/I387A mutation is caused by the promotion of ceramide\nsynthesis, this strain should also show resistance to myriocin, which inhibits the first\nstep of sphingolipid synthesis. Conversely, if decrease in ceramide levels due to\nLac1L379A/L383A/I387A mutation is the cause of aureobasidin A resistance, the\nstrain should show sensitivity to myriocin. The author could test this. In addition to the\ntest, I recommend to analyze not only changes in total ceramide levels but also\nchanges in ceramide species composition. It has been shown that changes in the\nchain length of ceramide fatty acids or the number of hydroxyl groups have impacts\non sensitivity to aureobasidin A (Tani and Kuge, J Biochem., 2010; Epstein et al., Mol.\nMicrobiol., 2012; Murakami et al., Sci. Rep., 2015). It is possible that\nLac1L379A/L383A/I387A mutation may influence substrate specificity for ceramide\nsynthase and the Lac1L379A/L383A/I387A mutant strain may produce specific\nceramide species. Therefore, examining if the Lac1L379A/L383A/I387A mutation\naffects ceramide levels and composition is very important and essential to support the\nmodel proposed by the authors. For this purpose, the authors can analyze lipidomics\nfor ceramide content and species in wild-type and mutant cells using mass\nspectrometry. Alternatively, the authors could measure in vitro ceramide synthase\nactivity using acyl-CoA with different lengths (from C14-CoA to C26-CoA) and not only\nDHS (dihydrosphinosine) but also PHS (phtosphingosine).\nWe thank the reviewer for raising these critical points and for the insightful\nsuggestions to strengthen our study. In the revised manuscript, we have\nconducted the recommended lipidomic analysis, which provides important new\ninsights into the functional role of the Lac1 dimerization interface.\nFollowing the reviewer’s suggestion, we performed lipidomic profiling of\nLac1WTΔLag1 and Lac1L379A/L383A/I387AΔLag1 cells after AbA treatment.\nSurprisingly, the Lac1 mutant cells accumulated significantly less ceramide than\nWT cells, particularly ceramide species bearing a C26 fatty acyl chain, which\nrepresents the major acyl-CoA substrate for Lac1 (Fig. 4D, table S2). In contrast,\ninositol phosphorylceramide (IPC) levels were comparable between WT and\nmutant cells (fig. S7A, B, table S2). These findings indicate that the AbA\nresistance phenotype of the Lac1L379A/L383A/I387A mutant arises primarily from\nreduced accumulation of very long-chain fatty acid (VLCFA)-containing\nceramides, rather than from increased synthesis of complex sphingolipids.\nNotably, a prior study demonstrated that defects in VLCFA biosynthesis confer\nresistance to growth inhibition induced by AUR1 repression in yeast (Tani and\nKuge, J Biochem., 2010). Together with our data, these observations suggest that\nthe Lac1L379A/L383A/I387A mutant causes the decrease in VLCFA-containing\nceramide production, thereby conferring AbA resistance.\nThis unexpected finding—disruption of the putative autoinhibitory interface\nreduces rather than increases ceramide accumulation—challenges a simple\nautoinhibition model. Since elevated ceramide levels are generally associated\nwith growth impairment, the more severe growth defect of Lac1WT cells under\nAbA stress (Fig. 4B) is consistent with their higher ceramide accumulation. The\npartial growth rescue of the interface mutant, despite lower ceramide levels,\nsuggests that this interface modulates the relationship between ceramide\nhomeostasis and cellular fitness under sphingolipid pathway stress.\nWhile the precise mechanism linking interface disruption to reduced ceramide\noutput remains to be elucidated, our data unequivocally demonstrate that the\nLac1-mediated dimerization interface plays a critical regulatory role in vivo,\ncontrolling both ceramide accumulation and cellular adaptation during complex\nsphingolipid depletion.\nThese new findings have led us to revise several key sections of the manuscript\nto reflect the updated interpretation:\n In the Abstract, we now state that the interface “is required for the\nregulation of ceramide output and cellular adaption during perturbation\nof complex sphingolipid biosynthesis,” moving beyond a simple\nautoinhibition narrative.\n In the Introduction, we have adjusted the final paragraph to clarify that\nthe interface does not merely act as an “off-switch,” but is essential for\nproper ceramide regulation under stress.\n In the Results section describing in vivo functional analysis, we have\nincorporated the lipidomics data (Fig. 4D, fig. S7A, B, and table S2) and\nexplicitly note that the mutation reduces ceramide accumulation,\nchallenging a straightforward autoinhibition model.\n In the Discussion, we have revised the opening paragraph to highlight\nthe apparent paradox between the structural evidence for autoinhibition\nand the reduced ceramide accumulation observed in vivo, stating that\n“the physiological function of this interface extends beyond being a\nsimple static inhibitor in vivo” and emphasizing the need to consider\nceramide turnover, subcellular distribution, and downstream integration\nin interpreting the lipidomics data. In the concluding paragraph, we\nhighlight the need for further investigations to elucidate the mechanistic\nbasis underlying this seemingly paradoxical behavior.\n Based on the latest lipidomics data and the aforementioned explanations,\nwe have revised the original title to \"Structural and functional dissection\nof a higher-order oligomerization interface in yeast ceramide synthase.\"\nWe have incorporated these new lipidomic data and corresponding\ninterpretations throughout the revised manuscript, and we believe they\nsignificantly strengthen the physiological relevance and mechanistic nuance of\nour findings.\nRegarding the reviewer’s suggestion to test myriocin sensitivity: we did perform\nthis experiment and observed that the Lac1 mutant and WT strains showed\ncomparable sensitivity (data shown below). Since this result does not align\nclearly with either of the two straightforward predictions proposed by the\nreviewer, and its mechanistic basis remains unclear, we have opted not to\ninclude it in the manuscript to avoid undue speculation. We agree that further\nstudies will be needed to fully understand how the interface mutation influences\npathway flux and stress responses.\nOther point:\nDo Lac13A and Lac13E mutations, which exhibit diminished ceramide synthase\nactivity, confer aureobasidin A resistance? If so, it is suggested that aureobasidin A\nresistance is due to reduced ceramide content in the mutant strains. How about\nsensitivity to myriocin?\nWe thank the reviewer for this insightful suggestion. As suggested, we assayed\nthe AbA and myriocin sensitivity of Lac13A and Lac13E (data shown below).\nLac13A grew poorly even on YPD (consistent with Fresques et al., JBC 2015),\nconfirming that severely impaired CerS activity is lethal. Lac13E was\nhypersensitive to both drugs; its AbA hypersensitivity stands in direct opposition\nto the AbA resistance of Lac1L379A/L383A/I387A.\nOur new lipidomics data indicate that AbA resistance in the Lac1L379A/L383A/I387A\nmutant correlates with reduced ceramide accumulation, rather than increased\noutput. This suggests that in vivo growth phenotypes cannot be predicted\nsimply by in vitro catalytic activity, likely due to the complex cellular environment\ninvolving ceramide turnover, trafficking, and metabolic integration. Further\nstudies will be required to dissect how each mutation distinctly rewires\nsphingolipid homeostasis and stress adaptation in cells.\nReviewer #3:\nQi Fang, Tian Xie, and Xin Gong provide “Structural insights into oligomerization-\ninduced autoinhibition of yeast.” They propose that a 4:4 oligomeric state of ceramide\nsynthase (CerS), formed by dimers of heterotetrameric 2:2 Lac1–Lip1 complexes,\nadopts an autoinhibited conformation. The structures of both the 2:2 Lac1–Lip1\nheterotetramer and the 1:2:1 Lac1–Lip1–Lag1 heterotetramer have recently been\ncharacterized by the authors and by several other groups. Overall, this is an\ninteresting finding, since oligomerization often autoinhibits the activity of enzymes\n(e.g., kinases such as TORC1).\nHowever, the manuscript does not provide sufficient evidence to support the idea that\nthe 4:4 CerS complex is indeed autoinhibited in vivo. I will not evaluate the structural\nanalysis, as this is outside my area of expertise.\nWe thank the reviewer for recognizing the significance of our findings and for\ntheir constructive feedback. We fully agree that establishing the physiological\nrelevance of the higher-order oligomerization state in vivo is essential. In\nresponse, we have performed key additional experiments as outlined in the\npoint-by-point responses below.\nMajor points:\n1. Is there evidence that endogenous 4:4 CerS complexes exist in vivo? As it stands,\nthe 4:4 complex could simply be an artifact of purifying overexpressed complexes.\nWe thank the reviewer for raising this important point. To examine the\noligomeric states of the endogenous Lac1–Lip1 complex in vivo, we enriched\nthe endogenously expressed complex via anti-Flag immunoprecipitation from\nΔLag1 cells expressing Flag-tagged Lac1 at its native genomic locus, followed\nby cross-linking. Subsequent SDS-PAGE analysis revealed two discrete bands\nthat migrated at positions identical to the cross-linked 2:2 and 4:4 assemblies\nseen with overexpressed protein. These findings confirm that Lac1 and Lip1\nassemble into both 2:2 and 4:4 complexes in vivo, establishing that the 4:4 form\nis not an artifact of overexpression. This new data and corresponding text have\nbeen included in the revised manuscript (fig. S2D, lines 145–150).\n2. What are the in vivo consequences for sphingolipid metabolism and the\nphysiological relevance of forming 4:4 CerS complexes? Can cells use reversible\noligomerization to tune CerS activity in vivo? Do cells that lose this autoinhibition (e.g.,\ncells expressing the Lac1L379A/L383A/I387A mutant) generate more ceramides and\nconsequently more complex sphingolipids?\nWe thank the reviewer for raising these key questions. Our new lipidomics data\nprovide direct in vivo evidence that the 4:4 assembly serves a regulatory rather\nthan simply inhibitory role:\n1. Consequences of disrupting the 4:4 interface: The Lac1L379A/L383A/I387A\nmutant accumulates significantly less ceramide than Lac1WT cells,\nparticularly ceramide species bearing a C26 fatty acyl chain, which\nrepresents the major acyl-CoA substrate for Lac1 (Fig. 4D). In contrast,\ninositol phosphorylceramide (IPC) levels were comparable between WT\nand mutant cells (fig. S7A, B). These findings indicate that the AbA\nresistance phenotype of the Lac1L379A/L383A/I387A mutant arises primarily\nfrom reduced accumulation of very long-chain fatty acid (VLCFA)-\ncontaining ceramides, rather than from increased synthesis of complex\nsphingolipids. This indicates the 4:4 complex is required for modulating\nproper ceramide output during metabolic perturbation.\n2. Physiological relevance and reversibility: The coexistence of 2:2 and 4:4\ncomplexes in vivo, along with the mutant’s altered ceramide levels and\npartial growth rescue under stress (Fig. 4B), supports the view that\nreversible oligomerization can tune CerS activity in response to\nmetabolic demand, though the signals that shift the 2:2↔4:4 equilibrium\nremain to identified.\n3. Loss of autoinhibition does not increase ceramide synthesis: Contrary to\na simple autoinhibition model, disruption of the 4:4 interface reduces\nrather than increases ceramide accumulation, demonstrating that this\ninterface acts as a dynamic regulatory node essential for adaptive\nsphingolipid homeostasis.\nThese findings, now integrated throughout the revised manuscript, highlight the\nnuanced physiological role of CerS oligomerization beyond a binary on/off\nswitch.\n3. Are Lac1–Lip1–Lag1 heterotetramers also detected as higher-order oligomers?\nWe thank the reviewer for raising this point. To examine whether Lac1–Lip1–\nLag1 heterotetramers can also form higher‑order oligomers, we co‑expressed\nStrep‑tagged Lac1, Flag‑tagged Lag1, and untagged Lip1 in HEK 293F cells\nand purified the heterocomplex using tandem affinity purification. BN‑PAGE\nanalysis showed that the Lac1–Lip1–Lag1 complex similarly assembles into\nhigher-order species. These data and the corresponding description have been\nadded to the revised manuscript (fig. S2B, lines 134–138).\n4. In the in vivo experiment, the authors use a lag1Δ strain and express\nLac1L379A/L383A/I387A. This experiment should also be performed in a lac1Δ strain,\nwith Lac1L379A/L383A/I387A expressed under its endogenous promoter and\nterminator. Such a strain would also serve as an excellent control for the experiments\nmentioned above.\nWe thank the reviewer for this important point and apologize for not clarifying\nour strain construction strategy. Our experiments were indeed performed in the\nrequested endogenous background: because lac1Δlag1Δ is lethal, we first\ngenerated lac1Δ cells, then re-inserted Lag1WT-Flag or Lac1L379A/L383A/I387A-Flag\nat the native LAC1 locus, and finally deleted LAG1 in both backgrounds. Thus,\nall phenotypic comparisons reflect the amino-acid substitutions in an\nendogenous context; this is now stated explicitly in the revised Methods (lines\n494-495 and 567-571).\nMinor point:\nThe authors write:\n“we generated FLAG-tagged Lac1 in a targeted Lag1 knockdown strain (designated\nas Lac1^WTΔLag1^) and its corresponding Lac1^L379A/L383A/I387A^ mutant strain\n(designated as Lac1L379A/L383A/I387AΔLag1).”\nThis is surely a deletion strain, in which LAG1 was knocked out by homologous\nrecombination, rather than a knockdown.\nWe thank the reviewer for this correction. The term ‘knockdown’ has been\ncorrected to ‘deletion’ in the revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors provided extensive additional experiments, further strengthening the manuscript. The following minor additions would further strengthen the presented work:\nA) While the FRET limitation presented justifies alternative approaches, the authors should acknowledge the limitations of immunoprecipitation for unstable or dynamic higher-order assemblies. The presented data are convincing but likely do not represent the physiological quantitative ratio of the complexes in vivo.\nB) The authors could not identify clear assembly-switching stressors, but I appreciate the additional experiments. Further exploration of factors that impact oligomerization switches may be addressed in future work. The presence of apo Acb1 alone should have limited impact, as presented by the authors; however, they did not mention loading Acb1 with acyl-CoA prior to BN-PAGE.\nC) The authors should add clear exclusion criteria, such as CTF cutoff and astigmatism values in Å. Additionally, the cFAR value of 0.33 indicates anisotropy (cFAR < 0.5), which the authors should comment on.\nD) While not crucial, the authors should add a color key for the hydrophobicity scale in Fig. S5. An alternative would be to use the mlp function in ChimeraX.\nI recommend publication of this manuscript in Nature Communications.", "reviewer_2": "The authors adequately addressed all of my concerns in the revised version. Thus, I think the paper is now suitable for publication in Nature Communications.", "reviewer_3": "The authors have partially addressed my previous requests. Their functional studies indicate that the oligomerization interface of Lac1 is required for the proper regulation of CerS and its enzymatic activity (ceramide output). Specifically, the Lac1L379A/L383A/I387A-Lip1 mutant complex failed to form the higher-order Lac1–Lip1 complex, and cells expressing the mutant CerS showed reduced ceramide levels after AbA treatment.\nThis result is unexpected and contrasts with the in vitro data, where the activity of oligomeric CerS was lower than that of the Lac1L379A/L383A/I387A-Lip1 mutant complex.\nUnfortunately, the authors do not make sufficient effort to reconcile this discrepancy between the proposed auto-inhibitory model (which is well supported by structural data and in vitro enzymatic assays) and the in vivo significance of oligomerization, which appears to support a different model or suggests possible compensatory effects.\nAs it stands, the function and physiological relevance of oligomerization remain unresolved"}, "author_response": "Response to reviewers’ comments:\nReviewer #1:\nThe authors provided extensive additional experiments, further strengthening the\nmanuscript. The following minor additions would further strengthen the presented\nwork:\nA) While the FRET limitation presented justifies alternative approaches, the authors\nshould acknowledge the limitations of immunoprecipitation for unstable or dynamic\nhigher-order assemblies. The presented data are convincing but likely do not\nrepresent the physiological quantitative ratio of the complexes in vivo.\nWe thank the reviewer for this thoughtful comment. We agree that\nimmunoprecipitation from detergent-solubilized membranes may not fully\npreserve the native equilibrium between oligomeric states. Accordingly, we\nhave added the following sentences to the Results section (lines 151–154): \"We\nacknowledge that our immunoprecipitation-based analysis of endogenous\ncomplexes likely does not capture their true dynamic equilibrium or absolute\nstoichiometric ratio in vivo. Future studies will be required to quantitatively\nassess the native oligomeric landscape of yCerS.\"\nB) The authors could not identify clear assembly-switching stressors, but I appreciate\nthe additional experiments. Further exploration of factors that impact oligomerization\nswitches may be addressed in future work. The presence of apo Acb1 alone should\nhave limited impact, as presented by the authors; however, they did not mention\nloading Acb1 with acyl-CoA prior to BN-PAGE.\nWe thank the reviewer for this valuable observation. We agree that acyl-CoA\nbinding may influence Acb1 conformation or its interaction with the CerS\ncomplex. To address this, we repeated the BN-PAGE analysis using Acb1 pre-\nloaded with C26-CoA. The results were comparable to those obtained with apo\nAcb1 (see figure below), indicating that acyl-CoA loading does not substantially\nalter the oligomeric states of the CerS complex under our experimental\nconditions.\nC) The authors should add clear exclusion criteria, such as CTF cutoff and\nastigmatism values in Å. Additionally, the cFAR value of 0.33 indicates anisotropy\n(cFAR < 0.5), which the authors should comment on.\nWe appreciate the reviewer's attention to these technical details. We have now\nexplicitly stated our CTF exclusion criteria in the Methods section (lines 566-\n567): \"The collected micrographs were curated and selected using a CTF\nresolution cutoff of 4 Å and an astigmatism threshold of less than 500 Å.\"\nRegarding the cFAR value, we have added the following sentence to the\nResults section (lines 174-178): \"Although the map exhibits a slight\nanisotropy—as indicated by a cFAR value of 0.33—the overall reconstruction\nof the 4:4 Lac1-Lip1 complex, including the dimeric interface between Lac1\nsubunits, is well-resolved and structurally coherent. Consequently, the higher-\norder 4:4 assembly and the conformational details of the Lac1 subunits at the\ndimeric interface are accurately determined (fig. S4A).\"\nD) While not crucial, the authors should add a color key for the hydrophobicity scale\nin Fig. S5. An alternative would be to use the mlp function in ChimeraX.\nWe thank the reviewer for this helpful suggestion. We have now added a color\nkey for the hydrophobicity scale to fig. S5 in the revised manuscript.\nReviewer #2:\nThe authors adequately addressed all of my concerns in the revised version. Thus, I\nthink the paper is now suitable for publication in Nature Communications.\nWe sincerely thank this reviewer for the positive assessment and for the\nconstructive feedback provided during the review process, which has helped us\nsubstantially improve the manuscript.\nReviewer #3:\nThe authors have partially addressed my previous requests. Their functional studies\nindicate that the oligomerization interface of Lac1 is required for the proper regulation\nof CerS and its enzymatic activity (ceramide output). Specifically, the\nLac1L379A/L383A/I387A-Lip1 mutant complex failed to form the higher-order Lac1-Lip1\ncomplex, and cells expressing the mutant CerS showed reduced ceramide levels\nafter AbA treatment.\nThis result is unexpected and contrasts with the in vitro data, where the activity of\noligomeric CerS was lower than that of the Lac1L379A/L383A/I387A-Lip1 mutant\ncomplex.\nUnfortunately, the authors do not make sufficient effort to reconcile this discrepancy\nbetween the proposed auto-inhibitory model (which is well supported by structural\ndata and in vitro enzymatic assays) and the in vivo significance of oligomerization,\nwhich appears to support a different model or suggests possible compensatory effects.\nAs it stands, the function and physiological relevance of oligomerization remain\nunresolved.\nWe thank the reviewer for raising this critical point. We fully agree that the\napparent discrepancy between our in vitro autoinhibition model and the in vivo\nfunctional outcome requires a more thorough and explicit reconciliation. We\nhave now substantially revised the manuscript to address this discrepancy. The\nkey additions are as follows:\n1. In the opening graph of Discussion, we have proposed the interface as a\nregulatory hub that couples ceramide synthesis to cellular demand, stress\nsignaling, or metabolic flux (lines 383–390): “We propose that the interface is\nnot merely a static 'off-switch' that dampens CerS activity when engaged.\nRather, it serves as a regulatory hub that couples ceramide synthesis to cellular\ndemand, stress signaling, or metabolic flux. In this view, the interface enables\ncontext-dependent tuning of CerS output, and its disruption uncouples the\nenzyme from these inputs, potentially resulting in inappropriately reduced\nceramide production under conditions that normally require sustained or\nelevated ceramide production. Thus, the paradoxical in vivo phenotype does\nnot invalidate the autoinhibitory mechanism but instead hints that the interface\noperates within a broader regulatory logic.”\n2. Building on the premise proposed in the original manuscript—that steady-\nstate ceramide accumulation does not directly reflect ceramide synthesis rates,\nas multiple pathways influence its final levels—we have further expanded the\nDiscussion to include several additional mechanisms that could potentially\naccount for the observed reduction in ceramide accumulation (lines 392-412):\n“Several possibilities, though speculative at this stage, could potentially account\nfor the reduced ceramide accumulation observed in the interface mutant under\nstress. One possibility relates to the complex metabolic fate of ceramides within\ncells. Given that cellular ceramide levels reflect not only synthesis but also\ndegradation, recycling, trafficking, and feedback regulation, the observed\ndecrease in steady-state ceramide accumulation could stem from increased\nceramide turnover, altered flux into downstream complex sphingolipids, or\nchanges in ceramide export or recycling—processes that are not captured by\nin vitro activity measurements alone. Alternatively, the interface could be\nrequired for proper subcellular localization, retention, or stability of the CerS\ncomplex under conditions of sphingolipid pathway perturbation. It is also\nconceivable that the interface facilitates productive interactions with upstream\nregulators such as Ypk1 or CK2; its disruption might impair this coordination,\nindirectly reducing ceramide accumulation through defective phosphorylation-\ndependent modulation. While purely speculative at this point, one might also\nconsider the possibility that the 4:4 assembly represents a \"primed\" or\n\"regulated\" state that, despite being partially inhibited, is poised for modulation\nin response to specific signals. Based on this hypothesis, disrupting this\ninterface could potentially lock the complex in an unregulated 2:2 state that fails\nto respond appropriately under stress, leading to dysregulated ceramide\nsynthesis. Distinguishing between these speculative models will require\nsystematic investigation of ceramide metabolic flux, enzyme turnover,\nsubcellular distribution, and protein-protein interaction networks in future\nstudies.”\n3. We have revised the concluding paragraph of the Discussion to more clearly\nunderscore the need to understand CerS regulation as an integrated, systems-\nlevel process and to highlight that the mechanistic basis for this paradox\nremains to be elucidated in future studies (line 472-474).\n4. The final sentence of the relevant Results subsection has also been revised\nto better frame the interpretive stance (lines 309-313): \"While the precise\nmechanism by which interface disruption leads to reduced ceramide\naccumulation under stress remains to be elucidated, these findings\nunequivocally demonstrate that this interface plays an essential regulatory role\nin vivo—one that extends beyond simple autoinhibition and is required for\nappropriate CerS output under conditions of sphingolipid pathway\nperturbation.\"\nWe believe these revisions substantially strengthen the manuscript by\nconfronting the apparent paradox, reframing it as a biologically meaningful\ninsight rather than an unresolved inconsistency, and providing a clear roadmap\nfor future mechanistic dissection. We thank the reviewer for pushing us to\narticulate this more clearly."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have adressed all raised concerns and comments, and I recommend publishing of their work.", "reviewer_3": "with the new text in the results and in the discussion, the authors have addressed my concerns."}, "author_response": ""}]

Decision letter