Elucidation of Lipid Asymmetry Signaling for the Prevention ofFungal Infections
Keisuke OBARA
Graduate School of Science, Nagoya University
Research objective
Deep-seated mycosis, in which pathogenic fungi penetrate deep into the organs, has a highmortality rate and poses a major threat to human health. Plant fungal infections, such as blast
disease and powdery mildew, have long been a threat to crops. Because infectious fungi areeukaryotes, chemical targets without side effects to humans are limited. Recently, the Rim101
pathway, which is activated by altered lipid asymmetry in the plasma membrane for adaptation,
has been reported to be required for the proliferation and virulence of fungi infecting their hostorganisms. We identified Rim21 as a sensor protein for alterations in lipid asymmetry in yeast
and we provide an overview of the Rim101 signaling pathway. Rim21 is considered an ideal
novel drug target because it regulates the Rim101 pathway at its most upstream position, andis exposed on the cell surface. A large black box in the Rim101 signaling pathway is themolecular mechanism by which Rim21 senses alterations in lipid asymmetry and outputs thesignal to downstream molecules. In this study, we aimed to elucidate this black box and form
a foundation for the development of drugs targeting Rim21.
Methods
It has been proposed that Rim21 uses its flexible C-terminal cytosolic tail region (Rim21C)
as an insect antenna to monitor lipid asymmetry through repetitive interactions with the innerleaflet of the plasma membrane1. Given this model, a recombinant protein containing theRim21C moiety was prepared and subjected to a lipid overlay assay to investigate Rim21Clipid interactions. We did not succeed in expressing GST-Rim21C in E. coli or mammaliancells; thus, we employed a cell-free expression system, that is, a wheat germ extract system.
We have previously reported that the ubiquitin ligase Rsp5 is essential for the Rim101
signaling pathway2. Ubiquitination of the sensor protein, Rim21, was examined using a pulldown assay of Rim21-HA from cells expressing His-tagged ubiquitin. Comprehensive
analysis showed that the K363 residue of Rim21 was ubiquitinated. Given this information,
we constructed a RIM21(K363R) mutant strain and monitored the progression of the Rim101
pathway and the ubiquitination of Rim21.
Rsp5 has three substrate-recognition domains, the so-called WW domains. Mutations wereintroduced into each WW domain and activation of the Rim101 pathway was examined. Many
Rsp5 substrates contain PxxY motifs, to which the WW domain of Rsp5 binds. Rim21 contains
a PxxY-like motif in the Rim21C moiety. The Rim101 pathway was monitored in cells
harboring mutations in the PxxY-like motif of Rim21.
The Rim21C moiety was cloned from the C. glabrata genome and expressed in S. cerevisiae
cells as a fusion protein with GFP. The response of GFP-CgRim21C to altered lipid asymmetry
was also observed.
ResultsRecombinant

GST-Rim21C

protein,prepared using the cell-free wheat germ
extract system, was subjected to a lipid overlay
assay. GST-Rim21C bound to acidic lipidspecies,
suchasphosphatidylserine,phosphatidylinositol, phosphatidic acid, andphosphatidylinositol(4,5)bis-phosphate (Fig.
1). This result is consistent with our previous
report that basic residues in the Rim21Cmoiety are required for plasma membranebinding, whereas a cluster of acidic residues isresponsible for repulsion from the membrane1. Phosphatidylethanolamine does not have acharge, but it mostly localizes to the inner leaflet of the plasma membrane and is, thus, one of
the lipids showing an asymmetric distribution. Phosphatidylethanolamine was then mixed
with the lipids to which GST-Rim21C bound, and a lipid overlay assay was performed. Of
note, binding of GST-Rim21C to acidic lipids was enhanced in the presence ofphosphatidylethanolamine (Fig. 1, right panel). Phosphatidylethanolamine has an amino groupthat accepts protons. We hypothesized that the equilibrium of the acidic lipids is tilted to anegatively charged state in the presence of phosphatidylethanolamine.
Ubiquitinated proteins were pulled down from the membrane fraction of cells expressing
His-tagged ubiquitin. Subsequent immunoblotting analysis revealed that Rim21-HA waspolyubiquitinated. Ubiquitination of Rim21-HA was facilitated by activation of the Rim101
pathway. Ubiquitination of Rim21-HA in the logarithmic growth phase was mostly abolishedby introducing the K363R mutation into Rim21-HA. However, the ubiquitination of
Rim21(K363R)-HA was restored upon activation of the Rim101 pathway. Introducing
mutations into the PxxY-like motif reduced Rim21-HA ubiquitination. The ubiquitination of
Rim21-HA was nearly undetectable when both mutations were introduced (Fig. 2). The
Rim101 pathway progressed normally in cells expressing Rim21(K363R)-HA. In contrast,

activation of the Rim101 pathway wascompletely abolished in cells expressing
Rim21-HA harboring mutations in the PxxYlike motif.
Among the three WW domains of Rsp5, theintroduction of mutations into the third WW
domain strongly inhibited the Rim101 pathway.
We monitored the dynamics of GFPCgRim21C in S. cerevisiae cells. Similar toGFP-ScRim21C, GFP-CgRim21C bound to theplasma membrane when the Rim101 pathway
was inactive, but dissociated from the plasma
membrane in cells with disturbed lipid
asymmetry.
Conclusion
Rim21C bound to negatively charged lipid species, and this binding was enhanced byphosphatidylethanolamine. All bound lipids and phosphatidylethanolamine are mostly
localized to the inner leaflet of the plasma membrane; thus, such interactions may occur inliving cells as well. Rim21 can detect alterations in the negative charge on the inner leaflet
surface and can be activated in vivo.
Ubiquitination of Rim21 is facilitated by the Rim101 pathway. Rim21 undergoes
ubiquitination at an unknown position in addition to the known K363 residue. Ubiquitination
at an unknown residue, but not at K363, is likely important for the activation of the Rim101
pathway. The PxxY-like motif in Rim21 is involved in Rim21 ubiquitination and Rim101
pathway progression. The third WW domain of Rsp5 is involved in Rim101 signaling.
CgRim21C behaves in a manner similar to ScRim21C.
References
1) Nishino, K., Obara, K., and Kihara, A. (2015) The C-terminal cytosolic region ofRim21 senses alterations in plasma membrane lipid composition: Insights into sensing
mechanisms for plasma membrane lipid asymmetry. J. Biol. Chem. 290: 30797-30805.
2) Obara, K., and Kihara, A. (2014) Signaling events of the Rim101 pathway occur at theplasma membrane in a ubiquitination-dependent manner. Mol. Cell. Biol. 34: 35253534.