Functional analysis of a novel endo-O-mannosidase in budding
yeast, Saccharomyces cerevisiae
Hiroto HIRAYAMA
Glycometabolome Team, Systems Glycobiology Research Group,RIKEN
Research aims
Glycosylation of nascent proteins is one of the most common co- and post-translational
modifications. This modification is known to play a critical role in various biologicalprocesses, such as development, cell differentiation, signal transduction, cell-cellinteractions, and cancer metastasis; it is also known to improve the properties of donor
proteins (e.g., increasing stability and half-life). The biological pathways andphysiological properties of glycosylation have been extensively studied. By contrast, it
is known that free-formed nitrogen-linked-glycans, designated as free N-glycans (fNgs),
which are liberated from glycans on glycoproteins, are accumulated in the cytosol(1,2),
but the biological significance of the accumulation of fNgs is unclear. To gain insight
into the metabolism and accumulation of fNgs in the cytosol, we examined thegeneration and degradation of fNgs in various growth conditions in budding yeast cells(Saccharomyces cerevisiae). Unexpectedly, we found that in the mannose-containing
culture (YPMan), yeast cells generated novel free glycans derived from O-linked sugar
chains on the glycoproteins, suggesting that yeast cells possess a novelendo-O-mannosidase (EOM). We also found that a mutant defective in a transcription
repressor, Cyc8, exhibits excessive de-mannosylation of the glycoproteins, due to thehyper-activation of EOM, and shows a severe growth defect phenotype; this is due toexcessive de-mannosylation, suggesting that the EOM activity was strictly regulated byCyc8. Our goal for this study, therefore, was the identification of the gene encoding
EOM, through 1) global screening for genetic interactions with CYC8 and 2) deletion of
the whole non-essential gene. Our broader goal is to gain deeper insights into the
biological and physiological significance of the generation of free glycan derived fromO-glycans, designated as free O-glycans (fOGs).

Results
Construction of the doxycycline-dependent CYC8 shutdown strain(TetOff-cyc8-degron-DAmP)

Previously, we found that cyc8∆ cells showed severe growth defects in YPManculture, due to the EOM-dependent, excessive de-mannosylation of the cell wall
proteins (Figure 1A). Therefore, we sought to identify whole-gene mutants that suppress
the cell wall defect shown in cyc8∆ cells, because the genes encoding EOM should befound in the pool of suppressor mutants of cyc8∆. Specifically, for identification of thegenes suppressing the mannose-sensitive phenotype shown in cyc8∆, we decided toconstruct a series of double mutants in a cyc8∆ background. To do this, we had plannedto cross cyc8∆ mutants with all single gene deletion mutants to generate diploid cells
that would contain the heterozygous double mutation (Figure 1B); then, following
meiotic cell division, the resulting haploid cells possessing double mutations(CYC8/cyc8∆ XXX/xxx∆) would be isolated. Unfortunately, however, it is known thatheterozygous mutant cells that have mutations in the CYC8 gene cannot enter meiosis,
due to the aberrant expression of the genes involved in meiosis(3). To overcome thisobstacle, we tried to construct a strain that can shut down the expression of CYC8 in thepresence of doxycycline (Dox). Therefore, the CYC8 promoter on the gene sequence
was exchanged with a doxycycline-dependent shutdown promoter (tetOx7CYC8TATApromoter). In this construct, we also inserted a CL1 degron sequence(4) and a mutation
called DAmP (Decreased Abundance by mRNA Perturbation) (5) to avoid the leaky
expression of CYC8, even in the presence of doxycycline (Figure 1C). As shown in
Figure 1D, TetOff-cyc8-degron-DAmP strains showed the strongest growth defectphenotype in the presence of Dox on the mannose-containing media(Ca[-URA]+Man+Dox), and also exhibited normal spore formation and normal meiotic
division in the Dox-free condition (data not shown). This suggested that theTetOff-cyc8-degron-DAmP strain should be suitable for global screening for the genetic
interactions associated with CY8. The construction of the series of double mutants iscurrently in progress.
Excessive de-mannosylation disrupts the cell wall integrity pathway
Our previous study showed that the cyc8∆ mutant exhibits severe cell wall defectphenotypes (e.g., temperature, calcofluor-white [CFW] sensitivity, and Congo red [CR]sensitivity). Based on this fact, we suspected that one major cell wall integrity pathway,the MAPK pathway, would be impaired, owing to the excessive de-mannosylation of
the sensor protein for MAPK localized to the cell surface (Figure 2A). To test thishypothesis, we first tried to examine the phosphorylation status of one of the signaltransduction intermediators, Mpk1, using anti-phospho-Mpk1 antibodies. As shown in
Figure 2B, irrespective of the carbon source in the culture media, the MAPK pathway inwild type cells was activated by the addition of the cell wall stressor, CFW. By contrast,
the MAPK pathway in cyc8∆ cells was slightly, but constitutively, activated in YPManculture. We next examined the O-glycosylation status of Wsc1, a sensor protein for theMAPK pathway, from 0 to 6 h after exchanging the old media with fresh YPGlc orYPMan. Only when cyc8∆ cells were cultured with YPMan media, slight but significant
mobility shifts in the Wsc1-HA band were observed (Figure 2C; Low-Mw Wsc1-HA).
This mobility shift was due to the excessive de-mannosylation of the stem region, which
is located in the extracellular space and is highly mannosylated (Figure 3). We also
observed reductions of stable Wsc1-HA on the cell surface of cyc8∆ cells (data notshown). Taken together, these observations lead us to conclude that the EOM-dependent,
harsh de-mannosylation in cyc8∆ cells causes dysfunctions in the stress-sensing
capability of Wsc1. From these data, we proposed a model for the dysfunction of theMAPK pathway in cyc8∆ cells, as shown in Figure 3. In cyc8∆ cells cultured withYPMan, as a result of the under-glycosylation of Wsc1, unstable Wsc1 causes
dysfunctions in the MAPK pathway under stress conditions. Therefore, cyc8∆ cells
cultured with YPMan, display the severe cell wall defect phenotype.
Conclusion
Analysis of the phenotype of cyc8∆ not only revealed the regulatory mechanisms ofthe novel molecule endo-O-mannosidase, but also revealed a part of its physiologicalfunction. However, the details of the biological significance and molecular functions ofEOM-dependent fOG generation remain unclear. Our future studies, which will include
global screening for the gene encoding EOM, as well as for the genes involved in thegeneration and regulation of fOGs, will help to address these concerns.
References
1) Harada Y, Hirayama H, Suzuki T,(2015) Generation and degradation of free
asparagine-linked glycans. Cell Mol Life Sci. 72(13):2509–33
2) Hirayama H, Hosomi A, Suzuki T, (2014) Physiological and molecular functions of

the cytosolic peptide:N-glycanase. Semin Cell Dev Biol. 41:110–20,
3) Enyenihi AH, Saunders WS. (2003) Large-scale functional genomic analysis ofsporulation and meiosis in Saccharomyces cerevisiae. Genetics 163(1):47–54
4) Gilon T, Chomsky O, Kulka RG. (1998) Degradation signals for ubiquitin system
proteolysis in Saccharomyces cerevisiae. EMBO J. 15;17(10):2759–66.
5) Schuldiner M et al., (2005) Exploration of the function and organization of the yeast
early secretory pathway through an epistatic miniarray profile. Cell 4;123(3):507–19.

Figure 1. Construction of the
doxycycline-dependent
shutdown strain,
TetOff-cyc8-degron-DAmP.
(A) cyc8∆ cells show the cellwall defect phenotype in themannose-containing culture
condition, due to excessive
EOM activity. (B) Strategy forthe construction of ~4,000
double mutants in a cyc8∆
background, via mating andmeiosis. (C) Schematic
structures of TetOff-cyc8degron-DAmP. (D) Study of thegrowth phenotype of various
TetOff-cyc8 mutants via
spotting assay.

Figure 2. EOM-dependent,
excessive de-mannosylation of
Wsc1 disrupts the MAPK
pathway.
(A) The MAPK pathway
mediates cell wall integrity
under cell wall stress conditions.
(B) Activation status of theMAPK pathway, confirmed by
the phosphorylation of Mpk1,
using anti-phosphorylated
Mpk1 antibody (C) Glycosylation status of Wsc1 in wild type and cyc8∆ cells, in YPGlc or
YPMan culture media.

Figure 3. A model for theimpairment of the MAPK
pathway in cyc8∆ cells
cultured with mannose.