Doxorubicin Inhibits Phosphatidylserine Decarboxylase and Modifies Mitochondrial Membrane Composition in HeLa Cells.

Doxorubicin (DXR) is a drug widely used in chemotherapy. Its mode of action is based on its intercalation properties, involving the inhibition of topoisomerase II. However, few studies have reported the mitochondrial effects of DXR while investigating cardiac toxicity induced by the treatment, mostly in pediatric cases. Here, we demonstrate that DXR alters the mitochondrial membrane composition associated with bioenergetic impairment and cell death in human cancer cells. The remodeling of the mitochondrial membrane was explained by phosphatidylserine decarboxylase (PSD) inhibition by DXR. PSD catalyzes phosphatidylethanolamine (PE) synthesis from phosphatidylserine (PS), and DXR altered the PS/PE ratio in the mitochondrial membrane. Moreover, we observed that DXR localized to the mitochondrial compartment and drug uptake was rapid. Evaluation of other topoisomerase II inhibitors did not show any impact on the mitochondrial membrane composition, indicating that the DXR effect was specific. Therefore, our findings revealed a side molecular target for DXR and PSD, potentially involved in DXR anti-cancer properties and the associated toxicity.

Arabidopsis ketoacyl-CoA synthase 16 (KCS16) forms C36 /C38 acyl precursors for leaf trichome and pavement surface wax.

The aliphatic waxes sealing plant surfaces against environmental stress are generated by fatty acid elongase complexes, each containing a ß-ketoacyl-CoA synthase (KCS) enzyme that catalyses a crucial condensation forming a new C─C bond to extend the carbon backbone. The relatively high abundance of C35 and C37 alkanes derived from C36 and C38 acyl-CoAs in Arabidopsis leaf trichomes (relative to other epidermis cells) suggests differences in the elongation machineries of different epidermis cell types, possibly involving KCS16, a condensing enzyme expressed preferentially in trichomes. Here, KCS16 was found expressed primarily in Arabidopsis rosette leaves, flowers and siliques, and the corresponding protein was localized to the endoplasmic reticulum. The cuticular waxes on young leaves and isolated leaf trichomes of ksc16 loss-of-function mutants were depleted of C35 and C37 alkanes and alkenes, whereas expression of Arabidopsis KCS16 in yeast and ectopic overexpression in Arabidopsis resulted in accumulation of C36 and C38 fatty acid products. Taken together, our results show that KCS16 is the sole enzyme catalysing the elongation of C34 to C38 acyl-CoAs in Arabidopsis leaf trichomes and that it contributes to the formation of extra-long compounds in adjacent pavement cells.

ECERIFERUM2-LIKE proteins have unique biochemical and physiological functions in very-long-chain fatty acid elongation.

The extension of very-long-chain fatty acids (VLCFAs) for the synthesis of specialized apoplastic lipids requires unique biochemical machinery. Condensing enzymes catalyze the first reaction in fatty acid elongation and determine the chain length of fatty acids accepted and produced by the fatty acid elongation complex. Although necessary for the elongation of all VLCFAs, known condensing enzymes cannot efficiently synthesize VLCFAs longer than 28 carbons, despite the prevalence of C28 to C34 acyl lipids in cuticular wax and the pollen coat. The eceriferum2 (cer2) mutant of Arabidopsis (Arabidopsis thaliana) was previously shown to have a specific deficiency in cuticular waxes longer than 28 carbons, and heterologous expression of CER2 in yeast (Saccharomyces cerevisiae) demonstrated that it can modify the acyl chain length produced by a condensing enzyme from 28 to 30 carbon atoms. Here, we report the physiological functions and biochemical specificities of the CER2 homologs CER2-LIKE1 and CER2-LIKE2 by mutant analysis and heterologous expression in yeast. We demonstrate that all three CER2-LIKEs function with the same small subset of condensing enzymes, and that they have different effects on the substrate specificity of the same condensing enzyme. Finally, we show that the changes in acyl chain length caused by each CER2-LIKE protein are of substantial importance for cuticle formation and pollen coat function.

The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1.

Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population.

Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.

Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.

Very-long-chain fatty acids (VLCFA) are precursors for various lipids playing important physiological and structural roles in plants. Throughout plant tissues, VLCFA are present in multiple lipid classes essential for membrane homeostasis, and also stored in triacylglycerols. VLCFA and their derivatives are also highly abundant in lipid barriers, such as cuticular waxes in aerial epidermal cells and suberin monomers in roots. VLCFA are produced by the fatty acid elongase (FAE), which is an integral endoplasmic reticulum membrane multi-enzymatic complex consisting of four core enzymes. The 3-ketoacyl-CoA synthase (KCS) catalyzes the first reaction of the elongation and determines the chain-length substrate specificity of each elongation cycle, whereas the other three enzymes have broad substrate specificities and are shared by all FAE complexes. Consistent with the co-existence of multiple FAE complexes, performing sequential and/or parallel reactions to produce the broad chain-length-range of VLCFA found in plants, twenty-one KCS genes have been identified in the genome of Arabidopsis thaliana. Using CRISPR-Cas9 technology, we established an expression platform to reconstitute the different Arabidopsis FAE complexes in yeast. The VLCFA produced in these yeast strains were analyzed in detail to characterize the substrate specificity of all KCS candidates. Additionally, Arabidopsis candidate proteins were transiently expressed in Nicotiana benthamiana leaves to explore their activity and localization in planta. This work sheds light on the genetic and biochemical redundancy of fatty acid elongation in plants.

Evidence for specific interaction between the RhoGAP domain from the yeast Rgd1 protein and phosphoinositides.

The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.

Evidence for functional links between the Rgd1-Rho3 RhoGAP-GTPase module and Tos2, a protein involved in polarized growth in Saccharomyces cerevisiae.

The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Two-hybrid screening identified Tos2p as a candidate Rgd1p-binding protein. Further analyses confirmed that Tos2p binds to the RhoGAP Rgd1p through its C-terminal region. Both Tos2p and Rgd1p are localized to polarized growth sites during the cell cycle and associated with detergent-resistant membranes. We observed that TOS2 overexpression suppressed rgd1Δ sensitivity to a low pH. In the tos2Δ strain, the amount of GTP-bound Rho3p was increased, suggesting an influence of Tos2p on Rgd1p activity in vivo. We also showed a functional interaction between the TOS2 and the RHO3 genes: TOS2 overexpression partially suppressed the growth defect of rho3-V51 cells at a restrictive temperature. We propose that Tos2p, a protein involved in polarized growth and most probably associated with the plasma membrane, modulates the action of Rgd1p and Rho3p in S. cerevisiae.

The Candida albicans Rgd1 is a RhoGAP protein involved in the control of filamentous growth.

Rho proteins are essential regulators of polarized growth in eukaryotic cells. These proteins are down-regulated in vivo by specific Rho GTPase Activating Proteins (RhoGAP). We investigated the role of Rgd1 RhoGAP, encoded by the Candida albicans RGD1 gene. We demonstrated that CaCdc42, CaRho3 and CaRho4 proteins had an intrinsic GTPase activity and that CaRgd1 stimulates in vitro GTP hydrolysis of these GTPases. Deletion of RGD1 in C. albicans results in sensitivity to low pH as already described for rgd1Δ in Saccharomyces cerevisiae. The role of Rgd1 in survival at low pH is conserved in the two yeast species as the CaRGD1 gene complements the Scrgd1Δ sensitivity. By tagging the RhoGAP with GFP, we found that CaRgd1 is localized at the tip and cortex of growing cells and during cytokinesis at the septation sites in yeast and filamentous forms. We investigated the effect of CaRgd1 on the control of the polarized growth. Removing CaRGD1 alleles increased filamentous growth and cells lacking CaRgd1 presented longer germ tubes. Conversely, RGD1 overexpression restricted hyphae growth. Our results demonstrate that Rgd1 is critical for filamentous formation in C. albicans especially for filamentous elongation.

OPA1-related dominant optic atrophy is not strongly influenced by mitochondrial DNA background.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA) are the most frequent forms of hereditary optic neuropathies. LHON is associated with mitochondrial DNA (mtDNA) mutations whereas ADOA is mainly due to mutations in the OPA1 gene that encodes a mitochondrial protein involved in the mitochondrial inner membrane remodeling. A striking influence of mtDNA haplogroup J on LHON expression has been demonstrated and it has been recently suggested that this haplogroup could also influence ADOA expression. In this study, we have tested the influence of mtDNA backgrounds on OPA1 mutations. METHODS: To define the relationships between OPA1 mutations and mtDNA backgrounds, we determined the haplogroup affiliation of 41 French patients affected by OPA1-related ADOA by control-region sequencing and RFLP survey of their mtDNAs. RESULTS: The comparison between patient and reference populations did not revealed any significant difference. CONCLUSION: Our results argue against a strong influence of mtDNA background on ADOA expression. These data allow to conclude that OPA1 could be considered as a "severe mutation", directly responsible of the optic atrophy, whereas OPA1-negative ADOA and LHON mutations need an external factor(s) to express the pathology (i.e. synergistic interaction with mitochondrial background).

New evidence of a mitochondrial genetic background paradox: impact of the J haplogroup on the A3243G mutation.

BACKGROUND: The A3243G mutation in the tRNALeu gene (UUR), is one of the most common pathogenic mitochondrial DNA (mtDNA) mutations in France, and is associated with highly variable and heterogeneous disease phenotypes. To define the relationships between the A3243G mutation and mtDNA backgrounds, we determined the haplogroup affiliation of 142 unrelated French patients - diagnosed as carriers of the A3243G mutation - by control-region sequencing and RFLP survey of their mtDNAs. RESULTS: The analysis revealed 111 different haplotypes encompassing all European haplogroups, indicating that the 3243 site might be a mutational hot spot. However, contrary to previous findings, we observed a statistically significant underepresentation of the A3243G mutation on haplogroup J in patients (p = 0.01, OR = 0.26, C.I. 95%: 0.08-0.83), suggesting that might be due to a strong negative selection at the embryo or germ line stages. CONCLUSION: Thus, our study supports the existence of mutational hotspot on mtDNA and a "haplogroup J paradox," a haplogroup that may increase the expression of mtDNA pathogenic mutations, but also be beneficial in certain environmental contexts.

Protein phosphorylation corrects the folding defect of the neuroblastoma (S120G) mutant of human nucleoside diphosphate kinase A/Nm23-H1.

Human nucleoside diphosphate (NDP) kinase A is a 'house-keeping' enzyme essential for the synthesis of nonadenine nucleoside (and deoxynucleoside) 5'-triphosphate. It is involved in complex cellular regulatory functions including the control of metastatic tumour dissemination. The mutation S120G has been identified in high-grade neuroblastomas. We have shown previously that this mutant has a folding defect: the urea-denatured protein could not refold in vitro. A molten globule folding intermediate accumulated, whereas the wild-type protein folded and associated into active hexamers. In the present study, we report that autophosphorylation of the protein corrected the folding defect. The phosphorylated S120G mutant NDP kinase, either autophosphorylated with ATP as donor, or chemically prosphorylated by phosphoramidate, refolded and associated quickly with high yield. Nucleotide binding had only a small effect. ADP and the non-hydrolysable ATP analogue 5'-adenyly-limido-diphosphate did not promote refolding. ATP-promoted refolding was strongly inhibited by ADP, indicating protein dephosphorylation. Our findings explain why the mutant enzyme is produced in mammalian cells and in Escherichia coli in a soluble form and is active, despite the folding defect of the S120G mutant observed in vitro. We generated an inactive mutant kinase by replacing the essential active-site histidine residue at position 118 with an asparagine residue, which abrogates the autophosphorylation. The double mutant H118N/S120G was expressed in inclusion bodies in E. coli. Its renaturation stops at a folding intermediate and cannot be reactivated by ATP in vitro. The transfection of cells with this double mutant might be a good model to study the cellular effects of folding intermediates.

Qualitative and quantitative study of the highly specialized lipid tissues of cetaceans using HR-MAS NMR and classical GC.

Cetacean adipose tissues contain an extremely very wide variety of acyl-chains present in triacylglycerols and / or wax esters. In addition, changes in the lipid composition across organs suggest fine stratification. It therefore remains technically challenging to describe precisely the lipid organization of these tissues. In the present study, we used in parallel HR-MAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) and GC (gas-chromatography) to characterize and quantify the lipids and fatty acyl-chains from the blubber and melon of two odontocete species. Both methods generated very similar compositions, but each presented clear advantages. While GC underestimated the amount of short branched fatty acyl-chains, which are specific to cetacean adipose tissues and most probably of primary importance for their functioning, HR-MAS NMR allowed for their exact quantification. Conversely, when HR-MAS NMR could only discriminate a few types of fatty acyl-chain families, GC unambiguously identified and quantified most of them. In addition, this technique allowed for the determination of the wax esters molecular species. Our results further suggest that the stratification of these adipose tissues relies on changes in the triacylglycerol to wax ester ratio and in the fatty acyl composition of triacylglycerols, but not on changes in the wax esters composition. Altogether, our data show that the complementarities of these two approaches result in lipid analyses of unprecedented precision, paving the way for the detailed description of the fatty acyl composition of cetacean adipose tissues and the understanding of their functioning.

RGD1, encoding a RhoGAP involved in low-pH survival, is an Msn2p/Msn4p regulated gene in Saccharomyces cerevisiae.

The RhoGAP Rgd1p is involved in different signal transduction pathways in Saccharomyces cerevisiae through its regulatory activity upon the Rho3 and Rho4 GTPases. The rgd1Delta mutant, which presents a mortality at the entry into the stationary phase in minimal medium, is sensitive to medium acidification caused by biomass augmentation. We showed that low-pH shock leads to abnormal intracellular acidification of the rgd1Delta mutant. Transcriptional regulation of RGD1 was studied in several stress conditions and we observed an activation of RGD1 transcription at low pH and after heat and oxidative shocks. The transcription level at low pH and after heat shock was demonstrated to depend on the STRE box located in the RGD1 promoter. The general stress-activated transcription factors Msn2p and Msn4p as well as the HOG pathway were shown to mainly act on the basal RGD1 transcriptional level in normal and stress conditions.

Mutation rate switch inside Eurasian mitochondrial haplogroups: impact of selection and consequences for dating settlement in Europe.

R-lineage mitochondrial DNA represents over 90% of the European population and is significantly present all around the planet (North Africa, Asia, Oceania, and America). This lineage played a major role in migration "out of Africa" and colonization in Europe. In order to determine an accurate dating of the R lineage and its sublineages, we analyzed 1173 individuals and complete mtDNA sequences from Mitomap. This analysis revealed a new coalescence age for R at 54.500 years, as well as several limitations of standard dating methods, likely to lead to false interpretations. These findings highlight the association of a striking under-accumulation of synonymous mutations, an over-accumulation of non-synonymous mutations, and the phenotypic effect on haplogroup J. Consequently, haplogroup J is apparently not a Neolithic group but an older haplogroup (Paleolithic) that was subjected to an underestimated selective force. These findings also indicated an under-accumulation of synonymous and non-synonymous mutations localized on coding and non-coding (HVS1) sequences for haplogroup R0, which contains the major haplogroups H and V. These new dates are likely to impact the present colonization model for Europe and confirm the late glacial resettlement scenario.

Through its F-BAR and RhoGAP domains, Rgd1p acts in different polarized growth processes in budding yeast.

Protein domain architecture can be used to construct supramolecular structures, to carry out specific functions and to mediate signaling in prokaryotic and eukaryotic cells. The Rgd1p protein of budding yeast contains two domains with different functions in the cell: the F-BAR and RhoGAP domains. The F-BAR domain has been shown to interact with membrane phospholipids and is thought to induce or sense membrane curvature. The RhoGAP domain activates the GTP hydrolysis of two Rho GTPases, thereby regulating different cellular pathways. Specific molecular interactions with the F-BAR and RhoGAP domains, cell signaling and interplay between these domains may allow the Rgd1p protein to act in several different biological processes, all of which are required for polarized growth in yeast.

Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis.

Voltage-dependent anion channel (VDAC)1 is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that overexpression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 expression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-MIM crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-X(L), indicative of PTP operation. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

Phosphoinositides affect both the cellular distribution and activity of the F-BAR-containing RhoGAP Rgd1p in yeast.

Cell polarity is a key element of development in most eukaryotes. The Rho GTPase-activating protein Rgd1p positively regulates the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p contains an F-BAR domain at its N-terminal end in addition to its RhoGAP domain at its C-terminal end. We demonstrate here that phospholipids discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity on Rho4p. The central region of the protein contiguous to the F-BAR domain is required for this stimulation. The F-BAR region binds to phosphoinositides in vitro and also plays a key role in the localization of Rgd1p to the bud tip and neck during the cell cycle. Studies of heat-sensitive mutants lacking phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate suggested that Rgd1p initially binds to Golgi membranes via phosphatidylinositol 4-phosphate and is then transported to the plasma membrane, where it binds phosphatidylinositol 4,5-biphosphate. We demonstrate here the dual effects of phosphoinositides on a RhoGTPase-activating protein. Phosphoinositides both regulate the recruitment and trafficking of Rgd1p to membranes via the F-BAR domain and specifically stimulate GTPase-activating protein activity, consistent with functional interplay between lipids, RhoGAP, and its related GTPases in yeast growth.

An intracellular wave of cytochrome c propagates and precedes Bax redistribution during apoptosis.

Bax is considered to be pivotal in inducing cytochrome c release (CCR) from mitochondria during apoptosis. Indeed, Bax redistributes to the mitochondrial outer membrane (MOM) upon activation and forms homo-multimers that are capable of permeabilizing the MOM. Our attempts to image this sequence of events in single live cells resulted in unexpected observations. Bax redistribution exhibited two distinct components: an early minor redistribution that was silent in terms of homo-multimerization and a major late redistribution that was synchronous with the formation of Bax multimers, but that proceeded belatedly, i.e. only after caspase 3/7 (C3/7) had already been activated. Intriguingly, neither of these two components of redistribution correlated with CCR, which turned out to be spatially organized, propagating as a traveling wave at constant velocity. Strikingly, propagation of the CCR wave (1) preceded signs of in situ Bax conformational activation; (2) appeared to be independent of autocatalytic loops involving a positive feedback of either C3/7, Ca(2+) mobilization or mitochondrial permeability transition; and (3) was triggered by diffuse stimulation with the synthetic Bak activator BH3I-1 but then proceeded independently of Bak activation. Thus, the CCR wave not only questions the exact role of Bax redistribution in cell death, but also indicates the existence of yet unidentified positive-feedback loops that ensure a spatiotemporal control of apoptosis at the subcellular scale.

The Rho3 and Rho4 small GTPases interact functionally with Wsc1p, a cell surface sensor of the protein kinase C cell-integrity pathway in Saccharomyces cerevisiae.

Rgd1, a GTPase-activating protein, is the only known negative regulator of the Rho3 and Rho4 small GTPases in the yeast Saccharomyces cerevisiae. Rho3p and Rho4p are involved in regulating cell polarity by controlling polarized exocytosis. Co-inactivation of RGD1 and WSC1, which is a cell wall sensor-encoding gene, is lethal. Another plasma membrane sensor, Mid2p, is known to rescue the rgd1Deltawsc1Delta synthetic lethality. It has been proposed that Wsc1p and Mid2p act upstream of the protein kinase C (PKC) pathway to function as mechanosensors of cell wall stress. Analysis of the synthetic lethal phenomenon revealed that production of activated Rho3p and Rho4p leads to lethality in wsc1Delta cells. Inactivation of RHO3 or RHO4 was able to rescue the rgd1Deltawsc1Delta synthetic lethality, supporting the idea that the accumulation of GTP-bound Rho proteins, following loss of Rgd1p, is detrimental if the Wsc1 sensor is absent. In contrast, the genetic interaction between RGD1 and MID2 was not due to an accumulation of GTP-bound Rho proteins. It was proposed that simultaneous inactivation of RGD1 and WSC1 constitutively activates the PKC-mitogen-activated protein kinase (MAP kinase) pathway. Moreover, it was shown that the activity of this pathway was not involved in the synthetic lethal interaction, which suggests the existence of another mechanism. Consistent with this idea, it was found that perturbations in Rho3-mediated polarized exocytosis specifically impair the abundance and processing of Wsc1 and Mid2 proteins. Hence, it is proposed that Wsc1p participates in the regulation of a Rho3/4-dependent cellular mechanism, and that this is distinct from the role of Wsc1p in the PKC-MAP kinase pathway.