Turnover Regulation of the Rho GTPase Cdc42 by Heat Shock Protein Chaperones and the MAPK Pathway Scaffold Bem4

TLDR: Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the regulation of cell polarity and signal-dependent morphogenesis in eukaryotes as mentioned in this paper.

Abstract: All cells maintain an axis of polarity that directs the orientation of growth. Cell polarity can be reorganized during development and in response to extrinsic cues to produce new cell types. Rho GTPases are central regulators of cell polarity and signal-dependent cell differentiation. We show here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned over by members of the Heat Shock family of Proteins (HSPs). The Hsp40p chaperone, Ydj1p, was required for turnover of Cdc42p by the NEDD4 E3 ubiquitin ligase, Rsp5p, in the proteosome. Cdc42p turnover was regulated by HSPs at high temperatures, and in aging cells where the protein formed aggregates, implicating HSPs in Rho GTPase quality control. We also show that Cdc42pQ61L, which mimics the active (GTP-bound) conformation of the protein, was turned over at elevated levels by Ydj1p and Rsp5p. A turnover-defective version of Cdc42pQ61L led to multibudding phenotypes, implicating Cdc42 turnover in singularity in cell polarization. Cdc42p turnover also impacted MAP kinase pathway specificity. A pathway-specific scaffold, Bem4p, stabilized Cdc42p levels, which biased Cdc42p function in one MAPK pathway over another. Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the regulation of cell polarity and signal-dependent morphogenesis. Significance StatementRho GTPases are switch-like proteins that govern major decisions in cell polarity and signaling in eukaryotes. We elucidate here a pathway that turns over the yeast Rho GTPase Cdc42p, which is mediated by the heat-shock family of proteins (HSPs) and the NEDD4-type E3 ubiquitin ligase Rsp5p. This finding provides a way for HSPs to exert their widespread effects on morphogenetic responses, phenotypic plasticity, and signaling pathways. We also found that turnover of an active version of Cdc42p is critical for modulating cell polarity. Cdc42p turnover also impacted its function in a pathway specific setting, as stabilization of Cdc42p by Bem4p (SmgGDS-type scaffold) influenced the activity of a specific MAPK pathway. HSPs may regulate Rho GTPase turnover in many systems.

Figures

Figure 2. GTP-locked Cdc42p is rapidly degraded in a Ydj1p-, Rsp5p- and proteasomedependent manner. A) WT (PC538) cells expressing a plasmid containing GFP-Cdc42p (Cdc42, PC6454) were incubated in SD-URA media containing 25 µg/ml of CHX and analyzed by IB at the indicated time points. Numbers indicate the relative band intensity to GFP-Cdc42p levels at time 0. Top panel corresponds to longer exposure time of anti-GFP blot. B) Same as panel A except cells contain plasmid GFP-Cdc42pQ61L (Q61L, PC7458). C) Levels of GFP-Cdc42p (Cdc42, black, set to 1 for time zero) and GFP-Cdc42pQ61L (Q61L, orange, also set to 1 for time zero). Levels of GFP-Cdc42pQ61L at time zero is 5-fold lower than GFP-Cdc42p. Error bars represent the standard deviation (S.D.) among biological replicates (n=2). D) Fluorescence microscopy of WT cells expressing GFP-Cdc42p (WT) or GFP-Cdc42pQ61L (Q61L). Scale bar, 5 µm. E) Relative fluorescence intensity of cells expressing GFP-Cdc42p (Cdc42) or GFP-Cdc42pQ61L (Q61L). Error bars represent the standard deviation (S.D.) among biological replicates, n>25, asterisk p-value <0.01. F) Ubiquitin levels of GFP-Cdc42p and GFP-Cdc42pQ61L by Co-IPT. Input and precipitated lysates were blotted against anti-GFP and anti-ubiquitin antibodies. Ponceau S staining indicates control for protein levels. IPT GFP blot for GFP-Cdc42pQ61L corresponds to a longer exposure time to equal the amount of precipitated protein to GFP-Cdc42p. Same exposure time was applied and is showed in the Co-IPT blot for GFP-Cdc42pQ61L. Numbers indicate relative band intensity of CoIPT Ub compared to IPT GFP intensity. G) GFP-Cdc42pQ61L levels in WT (PC3288) and rsp5-1 (PC3290) cells at the permissive temperature, 30°C. Numbers indicate relative levels of GFPCdc42p. H) GFP-Cdc42pQ61L levels in WT (S288c background, PC986) cells and ydj1D (PC7657). Numbers indicate the relative levels of GFP-Cdc42p compared to Pgk1p at time zero. I) WT cells (PC538) containing GFP-Cdc42pQ61L supplemented with 0.5% ethanol (control) or 75µM MG132 were incubated for 2 h. Numbers indicate relative levels of GFP-Cdc42p compared to Pgk1p.

Figure 4. Role of Cdc42p turnover in viability and singularity in polarization. A) Top, cdc42D strain harboring the pRS316-GFP-Cdc42 (URA3) and pRS315-GFP-Cdc42 (LEU2) or pRS315GFP-Cdc42TD (TD, K5,94,96R; LEU2) were examined for growth on SD-URA-LEU and 5-FOA media. Bottom, morphology of cdc42D strain harboring the pRS316-GFP-Cdc42 (URA3) and pRS315-GFP-Cdc42 (LEU2) or pRS315-GFP-Cdc42TD (TD, K5,94,96R; LEU2) after 2 days on 5-

Figure 6. Cdc42p turnover regulation by HSPs and Rsp5p impacts quality control, as well as singularity in polarization, MAPK signaling and polarity rejuvenation. A) Cdc42p turnover occurs by HSPs as part of a quality-control mechanism. Elevated temperatures (Heat) stimulate Cdc42p turnover, which occurs through Ydj1p, Rsp5p, and the proteasome. Overexpression of the protein, or elevated levels resulting from failure of the protein to be turned over, such as in cells lacking Ydj1p, lead to aggregation of the protein that can stimulate turnover. Aggregation can result from mutations or protein damage (star) that leads to instability. Such problems can accrue during cellular aging; hence, we speculate that the turnover of mis-folded Cdc42p can promote the overall ‘polarity health’ of cells and lead to the rejuvenation of cell polarity. Question mark, aspects of the turnover of Cdc42p aggregates are not clear. B) Cdc42p is activated by GEFs and inactivated by GAPs, which is thought to occur rapidly (FAST). Here we show that yeast Cdc42p is turned over by a mechanism that involves the HSP chaperone Ydj1p and the E3 ubiquitin ligase Rsp5p, which is slower than GAP hydrolysis (SLOW). Ubiquitin conjugates of Cdc42p (large red dots) results in degradation of the protein in the proteasome. Conformational changes that occur to the protein when activated by binding to GTP may lead to accelerated turnover of the protein by recognition by Ydj1p and/or Rsp5p. Turnover of Cdc42p promotes singularity in polarization, and is expected to attenuate MAPK pathway signaling (block arrow). Bem4p is a Cdc42p-interacting protein that may function to stabilize Cdc42p (red arrow). As a result, one MAPK pathway is stimulated among other Cdc42p-dependent MAPK pathways in the cell.

Figure 3. Lysine residues are required for Cdc42p turnover and stability. Alterations of Cdc42p lead to the formation of aggregates in mother cells. A) The yeast Cdc42p protein sequence was overlaid onto the crystal structure of human Cdc42p using the Expasy web server SWISS-MODEL (https://swissmodel.expasy.org) (Nassar et al., 1998). Yellow refers to lysines

Figure 1. Cdc42p is degraded at 37°C in a Hsp40p-, Hsp70p-, Rsp5p-, and proteasomedependent manner. A) Wild-type (WT) cells (S1278b background, PC538) were incubated at 30˚C in SD+AA media supplemented with 25 µg/ml of CHX and analyzed by IB at the indicated time points. Numbers indicate relative values of Cdc42p compared to Pgk1p at time zero. B) The same cells in panel A were incubated at 37˚C for the indicated time points and analyzed by IB. C) Cdc42p levels relative to Pgk1p at 30°C (black) and at 37°C (yellow). Error bars represent the standard deviation (S.D.) among biological replicates (n=2). D) Ubiquitination of Cdc42p was compared in lysates prepared from cells with (+) and without (-) the pGFP-Cdc42p plasmid by CoIPT. Input and precipitated lysates were blotted against anti-GFP and anti-ubiquitin (Ub) antibodies. Ponceau S staining was used in the Input lysates as a control for protein levels. Numbers indicate relative band intensity of Co-IPT Ub compared to the IPT GFP protein intensity. E) Levels of GFP-Cdc42p expressed in WT (S288c background, PC3288) and rsp5-1 (PC3290) cells grown for 4 h at 30°C and shifted to 37°C. Samples were analyzed at the indicated times. Numbers indicate relative GFP-Cdc42p levels compared to Pgk1p at time zero in WT. F) WT (PC5851) and cim3-1 (PC5852) cells expressing GFP-Cdc42p were grown at 37°C and analyzed by IB at the indicated time points. Numbers indicate the relative levels of GFP-Cdc42p compared to Pgk1p at time zero in WT. G) Fluorescence microscopy of WT cells (PC5851) expressing GFP-Cdc42p at 30°C or 37°C and cim3-1 (PC5852) cells grown at 37°C. Scale bar, 5 µm. H) Relative fluorescence intensity of GFP-Cdc42p expressed in cells described in panel G. Error bars represent the standard deviation (S.D.) among biological replicates, n>10, asterisk p-value <0.01. I) Cdc42p levels in WT cells

Figure 5. Turnover of Cdc42p differentially impacts MAPK pathway signaling: role of the scaffold Bem4p in stabilization of the Cdc42p protein. A) The model represents two Cdc42pdepenedent MAP kinase pathways. Colored text refers to fMAPK- (red) and mating- (yellow) specific components. Grey, common or shared factors. How Bem4p induces a pathway-specific signal at the level of Cdc42p remains unclear (question mark). B) Top, WT cells and ste11D expressing GFP-Cdc42pQ61L (Q61L) or GFP-Cdc42pQ61L,K5,94,96R (Q61L+TD). Bottom, halo formation in response to a-factor of WT cells expressing pGFP-Cdc42pQ61L (Q61L) or pGFPCdc42Q61L+TD (Q61L+TD). Cells were spotted onto SD-URA media, and a-factor was spotted at two concentrations, 6 µM or 2 µM. C) WT cells expressing GFP-Cdc42p (Cdc42), GFPCdc42pQ61L (Q61L) or GFP-Cdc42pQ61L,K5,94,96R (Q61L+TD) were grown for 6 h in SD-URA media. Numbers indicate relative P~Kss1p, P~Fus3p and GFP-Cdc42p compared to Pgk1p. D) Diagram of Cdc42p-interacting proteins. Colors indicate Cdc42p levels in extracts prepared from cells lacking the indicated protein. E) Cdc42p levels in WT cells and indicated mutants. Numbers indicate relative protein quantification. Cdc42p levels in cells lacking Gic2p and Bem4p were verified in multiple strains (Fig. S5A). F) Cdc42p levels in WT cells and bem1D , bem4D , ste11D mutants using antibodies to Cdc42p. Numbers indicate relative protein quantification. CDC42 mRNA levels were detected by RT-PCR. G) WT cells and bem4D mutant expressing GFP-Cdc42p were grown for 4 h at 30°C and examined by fluorescence microscopy. Scale bar, 5 µm. H) Relative fluorescence intensity of GFP-Cdc42p (Cdc42) or GFP-Cdc42pQ61L (Q61L) in WT cells and the bem4D mutant. n >25, asterisk p-value <0.01. I) Serial dilutions of WT and bem4D cells expressing

Full text

1
Turnover Regulation of the Rho GTPase Cdc42 by Heat Shock Protein
Chaperones and the MAPK Pathway Scaffold Bem4
Beatriz González and Paul J. Cullen
†
532 Cooke Hall
Department of Biological Sciences
State University of New York at Buffalo
Buffalo, NY 14260-1300
Phone: (716)-645-4923
FAX: (716)-645-2975
Corresponding author: †Paul J. Cullen
Email: pjcullen@buffalo.edu
Author Contributions: B.G. designed experiments, generated data and wrote the paper. P.J.C.
designed experiments and wrote the paper.
Competing Interest Statement: The authors don’t declare competing interest.
Keywords: cell polarity, protein quality control, scaffolds, MAPK signaling, protein aggregates
Abstract
All cells maintain an axis of polarity that directs the orientation of growth. Cell polarity can be
reorganized during development and in response to extrinsic cues to produce new cell types. Rho
GTPases are central regulators of cell polarity and signal-dependent cell differentiation. We show
here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned
over by members of the Heat Shock family of Proteins (HSPs). The Hsp40p chaperone, Ydj1p, was
required for turnover of Cdc42p by the NEDD4 E3 ubiquitin ligase, Rsp5p, in the proteosome.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2021. ; https://doi.org/10.1101/2021.07.13.452164doi: bioRxiv preprint

2
Cdc42p turnover was regulated by HSPs at high temperatures, and in aging cells where the protein
formed aggregates, implicating HSPs in Rho GTPase quality control. We also show that
Cdc42p
Q61L
, which mimics the active (GTP-bound) conformation of the protein, was turned over at
elevated levels by Ydj1p and Rsp5p. A turnover-defective version of Cdc42p
Q61L
led to
multibudding phenotypes, implicating Cdc42 turnover in singularity in cell polarization. Cdc42p
turnover also impacted MAP kinase pathway specificity. A pathway-specific scaffold, Bem4p,
stabilized Cdc42p levels, which biased Cdc42p function in one MAPK pathway over another.
Turnover regulation of Rho GTPases by HSPs and scaffolds provides new dimensions to the
regulation of cell polarity and signal-dependent morphogenesis.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2021. ; https://doi.org/10.1101/2021.07.13.452164doi: bioRxiv preprint

3
Significance Statement
Rho GTPases are switch-like proteins that govern major decisions in cell polarity and signaling in
eukaryotes. We elucidate here a pathway that turns over the yeast Rho GTPase Cdc42p, which is
mediated by the heat-shock family of proteins (HSPs) and the NEDD4-type E3 ubiquitin ligase
Rsp5p. This finding provides a way for HSPs to exert their widespread effects on morphogenetic
responses, phenotypic plasticity, and signaling pathways. We also found that turnover of an active
version of Cdc42p is critical for modulating cell polarity. Cdc42p turnover also impacted its
function in a pathway specific setting, as stabilization of Cdc42p by Bem4p (SmgGDS-type
scaffold) influenced the activity of a specific MAPK pathway. HSPs may regulate Rho GTPase
turnover in many systems.
.CC-BY-NC-ND 4.0 International licenseavailable under a
was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint (whichthis version posted July 14, 2021. ; https://doi.org/10.1101/2021.07.13.452164doi: bioRxiv preprint

4
Main Text
Introduction
Cells establish an axis of polarity to maintain cell shape and to orient cell growth and division. Cell
polarity can be reorganized in response to extrinsic cues to impact many biological processes,
including cell motility (chemotaxis/chemotropism), development, and differentiation to specific
cell types. Ras homology (Rho) GTPases are master regulators of cell polarity and signaling
(Etienne-Manneville and Hall, 2002). Rho GTPases cycle between an active GTP-bound
conformation that is regulated by guanine nucleotide exchange factors (GEFs), and an inactive
GDP-bound conformation this is regulated by GTPase-activating proteins (GAPs). In the GTP-
bound conformation, Rho GTPases interact with effector proteins to regulate cellular responses
through the reorganization of cytoskeletal elements (Sit and Manser, 2011), the induction of
Mitogen-Activated Protein Kinase (MAPK) pathways and other pathways (Coso et al., 1995; Van
Aelst and D'Souza-Schorey, 1997), by interaction with p21-activated (PAK) kinases (Ha and
Boggon, 2018; Rane and Minden, 2014; Tetley et al., 2017), and by other mechanisms. Rho
GTPases are also regulated by guanine nucleotide dissociation inhibitors [GDIs, (Boulter et al.,
2010; Hoffman et al., 2000)] and adaptor proteins (Irazoqui et al., 2003; Lamas et al., 2020) that
impact their function, localization, and activity. Rho GTPases can be modified by post-translational
modifications (Barthelmes et al., 2020) that include AMPylation (Barthelmes et al., 2020; Woolery
et al., 2014), phosphorylation (Chang et al., 2011; Ellerbroek et al., 2003), and ubiquitination
(Majolée et al., 2019). Ubiquitin-dependent turnover of Rho GTPases by E3 ubiquitin ligases
impacts Rho GTPase function in many settings (Goka and Lippman, 2015; Oberoi-Khanuja and
Rajalingam, 2012; Tian et al., 2011; Wang et al., 2003; Wei et al., 2013). Given the multitude of
functions regulated by Rho GTPases, an important open question is how Rho GTPases are directed
to specific responses in different settings. This question is relevant because mis-regulation of Rho
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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5
GTPase function is an underlying cause of cancer and other diseases (Haga and Ridley, 2016;
Svensmark and Brakebusch, 2019).
Heat Shock Proteins (HSPs) are evolutionarily conserved chaperones that induce protein
folding and also promote degradation of proteins that cannot be refolded (Balchin et al., 2016;
Whitesell and Lindquist, 2005). HSPs control a broad diversity of morphogenetic responses,
including the regulation of cell polarity, morphogenetic plasticity and signal transduction
(Calderwood and Gong, 2016; Grad et al., 2011; Pivovarova et al., 2007; Rutherford and Lindquist,
1998; Sun et al., 2021; Walsh et al., 1997; Whitesell and Lindquist, 2005). Despite the fact that
HSPs are widely considered to be evolutionary drivers of phenotypic plasticity, the mechanism by
which HSPs control cell polarity and signaling – and how they connect to and regulate cell polarity
machinery - remains in many cases unclear.
One of the best studied and most well understood Rho GTPases is the highly conserved
yeast Cdc42p [yeast and human Cdc42p are 81% identical (Bi and Park, 2012; Kozminski et al.,
2000)]. In yeast, Cdc42p is an essential protein and the master regulator of polarity establishment
(Bi and Park, 2012; Irazoqui and Lew, 2004; Pringle et al., 1995). Cdc42p also regulates multiple
MAPK pathways (Bardwell, 2005; Saito, 2010; Schwartz and Madhani, 2004) that induce different
morphogenetic responses through transcriptional regulation of non-overlapping sets of target
genes. Several well-defined mechanisms account for specification of shared components, between
MAPK pathways, including the utilization of scaffolds (Choi et al., 1994; Marcus et al., 1994;
Posas and Saito, 1997; Printen and Sprague, 1994) and degradation of pathway-specific factors
(Bao et al., 2004; Chou et al., 2004). However, it remains unclear how Cdc42p and other proteins
selectively regulate MAPK pathways that share components. In addition to these roles, Cdc42p is
a component of the exocyst complex (Adamo et al., 2001), which controls vesicle delivery to the
plasma membrane (Munson and Novick, 2006). Cdc42p also regulates the lysosome/vacuole (Jones
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Frequently asked questions

Q1. What are the contributions mentioned in the paper "Turnover regulation of the rho gtpase cdc42 by heat shock protein chaperones and the mapk pathway scaffold bem4" ?

The authors show here that one of the best understood Rho GTPases, the highly conserved yeast Cdc42p, is turned over by members of the Heat Shock family of Proteins ( HSPs ). CC-BY-NC-ND 4. 0 International license available under a was not certified by peer review ) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 

Q2. What is the role of Cdc42p in RhoGTPase?

Cdc42p aggregatespreferentially localized to mother cells, which implicates turnover regulation by HSPs in RhoGTPase quality control. 

Q3. What is the role of the HSPs in the turnover of Cdc42p?

It has been shownthat heat shock proteins (HSPs) can function as adaptor proteins that mediate the turnover ofproteins lacking PY motifs in an Rsp5p-dependent manner (Fang et al., 2014). 

Q4. What is the role of Cdc42p in morphogenetic responses?

Cdc42p also regulates multipleMAPK pathways (Bardwell, 2005; Saito, 2010; Schwartz and Madhani, 2004) that induce differentmorphogenetic responses through transcriptional regulation of non-overlapping sets of targetgenes. 

Q5. What is the role of bem4p in the fMAPK pathway?

Bem4p binds to other proteins to regulate the fMAPK pathway, including the GEF Cdc24pand the MAPKKK, Ste11p (Pitoniak et al., 2015). 

Q6. What is the role of Ydj1p in the regulation of Cdc42?

Ydj1p functions as a co-chaperone for Hsp70proteins (Becker et al., 1996), which also regulate protein degradation by the ubiquitin-proteasomesystem (Guerriero et al., 2013; Lee do et al., 2016). 

Q7. What is the way to keep misfolded proteins in the cell?

Aggregates of misfolded proteins can be kept in larger inclusionsin certain locations within the cell to prevent harmful interactions and facilitate degradation. 

Q8. What is the role of Cdc42p in the fMAPK pathway?

To determine the contribution that Bem4pdependent stabilization of Cdc42p plays on the activity of the fMAPK pathway, Cdc42pQ61L+TD was expressed in cells lacking Bem4p. 

Q9. What is the role of HSPs in morphogenetic responses?

HSPs control a broad diversity of morphogenetic responses,including the regulation of cell polarity, morphogenetic plasticity and signal transduction(Calderwood and Gong, 2016; Grad et al., 2011; Pivovarova et al., 2007; Rutherford and Lindquist,1998; Sun et al., 2021; Walsh et al., 1997; Whitesell and Lindquist, 2005). 

Q10. What is the role of Cdc42p in polarity establishment?

In yeast, Cdc42p is an essential protein and the master regulator of polarity establishment(Bi and Park, 2012; Irazoqui and Lew, 2004; Pringle et al., 1995). 

Q11. What is the role of rsp5p in the turnover of cytosolic?

Rsp5pregulates the turnover of cytosolic proteins in the proteasome (Brückner et al., 2011; Challa et al.,2021; Kowalski et al., 2018) and integral-membrane proteins by vesicular trafficking to thelysosome/vacuole (Katzmann et al., 2001; Lin et al., 2008). 

Q12. What is the effector of Cdc42p on bud growth?

TD induced more growth sites than wildtype, indicating that bud sites function to restrict growth from the accumulation of GTP-boundCdc42p (Fig. 4B). 

Q13. What is the effect of the cycloheximide on Cdc42p?

Consistent with thesefindings, the authors found that Cdc42p was a stable at 30˚C based on treatment with the protein synthesisinhibitor cycloheximide (Fig. 1A, CHX). 

Q14. What is the ubiquitination profile of GFP-Cdc42pQ?

strains containing plasmids expressing GFP-Cdc42pQ61L (Fig. S2A) and GFP-Cdc42pG12V (Fig. S2B), which both mimic theGTP-bound conformation of the protein, showed lower steady-state levels of Cdc42p. 

Q15. What is the effect of Cdc42p turnover on mating?

The formation of haloes of growth-arrested cells, which is induced by the mating pheromone a-factor, was not influenced by Cdc42pQ61L+TD(Fig. 5B, bottom panel). 

Q16. Why does Fus3p not activate the fMAPK pathway?

This may be because Fus3p exists in a conformationally inactive state in theabsence of pheromone and cannot be activated without binding to the scaffold Ste5p (Good et al., 2009).