ATPase Associated With Various Cellular Activities Research Articles

Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Clinical and genetic characteristics in a Chinese cohort of complex spastic paraplegia type 4.

Spastic paraplegia type 4 (SPG4), caused by SPAST mutations, is the most predominant subtype of hereditary spastic paraplegia. Most documented SPG4 patients present as pure form, with the complex form rarely reported. We described the clinical and genetic features of 20 patients with complex phenotypes of SPG4 and further explored the genotype-phenotype correlations. We collected detailed clinical data of all SPG4 patients and assessed their phenotypes. SPAST gene mutations were identified by Multiplex ligation-dependent probe amplification in combination with whole exome sequencing. We further performed statistical analysis in genotype and phenotype among patients with various manifestations and different variants. Out of 90 SPG4 patients, 20 patients (male:female = 16:4) with additional neurologic deficits, namely complex form, were included in our study. The bimodal distribution of age of onset at 0-10 and 21-40 years old is concluded. On cranial MRI, obvious white matter lesions can be observed in five patients. We identified 9 novel and 8 reported SPAST mutations, of which 11 mutations were located in AAA (ATPase associated with various cellular activities) domain. The AAA cassette of spastin is the hottest mutated region among complex SPG4.All patients with cognitive impairment (CI) are males (n = 9/9). Additionally, 80% patients with ataxia are due to frameshift mutations (n = 4/5). Overall, our study summarized and analyzed the genetic and phenotypic characteristics of complex SPG4, making up over 1/5 of in-house SPG4 cohort, among which CI and ataxia are the most common features. Further studies are expected to explore the underlying mechanisms.

Expression of recombinant mouse spastin in E. coli

Mutations of the gene SPAST that encodes a microtubule severing enzyme, spastin, are the most frequent cause of Hereditary spastic paraplegia (HSP) disease. HSP is heterogeneous group of inherited neurodegenerative disorders characterized predominantly by progressive lower limb spasticity and weakness. Spastin belongs ATPase associated with various cellular activities (AAA) protein family and catalyzes microtubule severing. Spastin in mouse and human are highly identical in protein sequence and several spastin mutation models in mice have been generated in order to evaluate the significance of spastin loss-of-function in mammals. Expression and purification of spastin and the mutant variants determined in patients will facilitate the structure-function relationship study of spastin. Here I systemically optimized the expression condition of a truncated version of mouse spastin in E. coli. The recombinant protein and a mutant were further purified for ATPase activity assay.

Structural basis for nucleotide-modulated p97 association with the ER membrane

Association of the cytosolic AAA (ATPases associated with various cellular activities) protein p97 to membranes is essential for various cellular processes including endoplasmic reticulum (ER)-associated degradation. The p97 consists of two ATPase domains and an N domain that interacts with numerous cofactors. The N domain of p97 is known to undergo a large nucleotide-dependent conformation switch, but its physiological relevance is unclear. Here we show p97 is recruited to canine ER membranes predominantly by interacting with VCP-interacting membrane protein (VIMP), an ER-resident protein. We found that the recruitment is modulated through a nucleotide-dependent conformation switch of the N domain in wild-type p97, but this modulation is absent in pathogenic mutants. We demonstrate the molecular mechanism of the modulation by a series of structures of p97, VIMP and their complexes and suggest a physiological role of the nucleotide-dependent N domain conformation switch. The lack of modulation in pathogenic mutants is caused by changes in interactions between the N and D1 domain, as demonstrated by multiple intermediate positions adopted by N domains of mutant p97. Our findings suggest the nucleotide-modulated membrane association may also have a role in other p97-dependent processes.

Escherichia coli ClpB is a non-processive polypeptide translocase.

Escherichia coli caseinolytic protease (Clp)B is a hexameric AAA+ [expanded superfamily of AAA (ATPase associated with various cellular activities)] enzyme that has the unique ability to catalyse protein disaggregation. Such enzymes are essential for proteome maintenance. Based on structural comparisons to homologous enzymes involved in ATP-dependent proteolysis and clever protein engineering strategies, it has been reported that ClpB translocates polypeptide through its axial channel. Using single-turnover fluorescence and anisotropy experiments we show that ClpB is a non-processive polypeptide translocase that catalyses disaggregation by taking one or two translocation steps followed by rapid dissociation. Using single-turnover FRET experiments we show that ClpB containing the IGL loop from ClpA does not translocate substrate through its axial channel and into ClpP for proteolytic degradation. Rather, ClpB containing the IGL loop dysregulates ClpP leading to non-specific proteolysis reminiscent of ADEP (acyldepsipeptide) dysregulation. Our results support a molecular mechanism where ClpB catalyses protein disaggregation by tugging and releasing exposed tails or loops.

Structure and Evolution of N-domains in AAA Metalloproteases

Metalloproteases of the AAA (ATPases associated with various cellular activities) family play a crucial role in protein quality control within the cytoplasmic membrane of bacteria and the inner membrane of eukaryotic organelles. These membrane-anchored hexameric enzymes are composed of an N-terminal domain with one or two transmembrane helices, a central AAA ATPase module, and a C-terminal Zn2+-dependent protease. While the latter two domains have been well studied, so far, little is known about the N-terminal regions. Here, in an extensive bioinformatic and structural analysis, we identified three major, non-homologous groups of N-domains in AAA metalloproteases. By far, the largest one is the FtsH-like group of bacteria and eukaryotic organelles. The other two groups are specific to Yme1: one found in plants, fungi, and basal metazoans and the other one found exclusively in animals. Using NMR and crystallography, we determined the subunit structure and hexameric assembly of Escherichia coli FtsH-N, exhibiting an unusual α+β fold, and the conserved part of fungal Yme1-N from Saccharomyces cerevisiae, revealing a tetratricopeptide repeat fold. Our bioinformatic analysis showed that, uniquely among these proteins, the N-domain of Yme1 from the cnidarian Hydra vulgaris contains both the tetratricopeptide repeat region seen in basal metazoans and a region of homology to the N-domains of animals. Thus, it is a modern-day representative of an intermediate in the evolution of animal Yme1 from basal eukaryotic precursors.

The Maize AAA-Type Protein SKD1 Confers Enhanced Salt and Drought Stress Tolerance in Transgenic Tobacco by Interacting with Lyst-Interacting Protein 5

ATPase associated with various cellular activities (AAA) proteins are important regulators involved in diverse cellular functions. To date, the molecular mechanisms of AAA proteins involved in response to salt and drought stresses in plants are largely unknown. In this study, a putative SKD1 (suppressor of K + transport growth defect 1) ortholog from Zea mays (ZmSKD1), which encodes a putative AAA protein, was isolated. The transcript levels of ZmSKD1 were higher in aerial tissues and were markedly up-regulated by salt or drought stress. Over-expression of ZmSKD1 in tobacco plants enhanced their tolerances not only to salt but to drought. Moreover, reactive oxygen species accumulations in ZmSKD1 transgenic lines were relative less than those in wild-type plants during salt or PEG-induced water stress. The interaction between ZmSKD1 and NtLIP5 (Lyst-Interacting Protein 5 homolog from Nicotiana tabacum) was confirmed by both yeast two-hybrid and immuno-precipitation assays; moreover, the α-helix-rich domain in the C-terminus of ZmSKD1 was identified to be required for its interaction with NtLIP5 using truncation mutations. Collectively, these data demonstrate that ZmSKD1could be involved in salt and drought stress responses and its over-expression enhances salt or drought stress tolerance possibly through interacting with LIP5 in tobacco. This study may facilitate our understandings of the biological roles of SKD1-mediated ESCRT pathway under stress conditions in higher plants and accelerate genetic improvement of crop plants tolerant to environmental stresses.

The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

ALG-2-interacting Tubby-like protein superfamily member PLSCR3 is secreted by an exosomal pathway and taken up by recipient cultured cells

PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca2+-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4BE235Q, a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP–Rab5AQ79L). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.

N-Terminal Phosphorylation of p60 Katanin Directly Regulates Microtubule Severing

Proteins of the AAA (ATPases associated with various cellular activities) family often have complex modes of regulation due to their central position in important cellular processes. p60 katanin, an AAA protein that severs and depolymerizes microtubules, is subject to multiple modes of regulation including a phosphorylation in the N-terminal domain involved in mitotic control of severing. Phosphorylation decreases severing activity in Xenopus egg extracts and is involved in controlling spindle length. Here, we show that the evolutionarily divergent N-terminal domains of p60 have maintained hotspots of mitotic kinase regulation. By reconstituting in vitro severing reactions, we show that phosphomimetic modification at amino acid position 131 in Xenopus laevis p60 decreases severing and microtubule-stimulated ATPase activity without affecting the binding affinity of p60 for microtubules. At high concentrations of the phosphomimetic mutant p60, wild-type levels of activity could be observed, indicating a more switch-like threshold of activity that is controlled by regulating oligomerization on the microtubule. This provides a cellular mechanism for high local concentrations of p60, like those found on spindle poles, to maintain severing activity while most of the protein is inhibited. Overall, we have shown that the modular domain architecture of AAA proteins allows for precise control of cellular activities with simple modifications.

Growing sphere of influence: Cdc48/p97 orchestrates ubiquitin-dependent extraction from chromatin

The AAA (ATPases associated with various cellular activities) family member Cdc48/p97 is best known for its role in ubiquitin-dependent proteasomal degradation of aberrant endoplasmic reticulum (ER) proteins, a process known as ER-associated degradation (ERAD). However, recent studies have also defined Cdc48/p97 as a central player in various chromatin-associated processes linked to cell cycle progression, DNA replication, transcription, and the DNA damage response. Notwithstanding the apparent differences in location and function, the role of Cdc48/p97 in ubiquitin-dependent extraction from chromatin (UDEC) bears striking similarities with its action in ERAD. Here, we discuss recent data that expand our current model of the role of Cdc48/p97 as a ubiquitin-selective segregase in the nuclear chromatin environment.

ATP-dependent assembly of the heteromeric Pex1p–Pex6p-complex of the peroxisomal matrix protein import machinery

The peroxisomal matrix protein import is facilitated by soluble receptor molecules which cycle between cytosol and the peroxisomal membrane. At the end of the receptor cycle, the import receptors are exported back to the cytosol in an ATP-dependent manner catalyzed by Pex1p and Pex6p, two AAA (ATPases associated with various cellular activities) type ATPases. Pex1p and Pex6p interact and form a heteromeric complex. In order to gain more insight into the stoichiometry and mechanism of assembly of the complex, we heterologously expressed and purified Saccharomyces cerevisiae Pex1p and Pex6p. Size exclusion chromatography studies of the recombinant proteins demonstrate that they form a hexameric complex in a one-to-one ratio of both AAA-proteins. The recombinant AAA-complex exhibits an ATPase activity with a k(m) of 0.17 mM and V(max) of 0.35 nmol min(-1) μg(-1). In the presence of N-ethylmaleimide, ATPase activity of the peroxisomal AAA-complex is drastically decreased and the complex dissociates. Disassembly of the complex into its Pex1p and Pex6p subunits is also observed upon ATP-depletion, indicating that formation of the Pex1p/Pex6p-complex requires the presence of ATP.

Effect of Ca2+ on the microtubule‐severing enzyme p60‐katanin. Insight into the substrate‐dependent activation mechanism

Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.

Simulations of the p97 complex suggest novel conformational states of hydrolysis intermediates

The vitally important AAA (ATPases associated with various cellular activities) protein p97 is involved in cellular functions ranging from replication to degradation of misfolded proteins and has recently been proposed as a novel chemotherapeutic target. p97 is a large molecular machine that has been shown to hexamerize in vitro, with each monomer consisting of an N domain responsible for binding to effector proteins and two AAA repeats (D1 and D2). However, structural studies are inconclusive or in disagreement with one another on several important features such as the locations of the N domains, the relative orientations of the D1 and D2 rings, and the dimensions of the central pore. Here, we present atomic-scale simulations of the p97 hexamer in the prehydrolysis, transition, and post-hydrolysis states. To improve the agreement between low- and high-resolution experimental studies, we first use a biased simulation technique, molecular dynamics flexible fitting (MDFF), to improve the correlation between the structures described in these experiments. We follow this with extended, classical molecular dynamics simulations, which not only show that structures generated in the MDFF phase are stable, but reveal insights into the dynamics important to each state. Simulation results suggest a hybrid model for hydrolysis, in which the N and D2 domains are dynamic while the D1 domains are relatively static, salt bridges stabilize the position of the N domains in the pre-hydrolysis state, and the rings formed by D1 and D2 rotate relative to one another.

FtsH proteases located in the plant chloroplast

FtsHs are a well-characterized family of membrane bound proteases containing an AAA (ATPase associated with various cellular activities) and a Zn(2+) metalloprotease domain. FtsH proteases are found in eubacteria, animals and plants and are known to have a crucial role in housekeeping proteolysis of membrane proteins. In Arabidopsis thaliana, 12 FtsH family members are present (FtsH 1-12) and their subcellular localization is restricted to mitochondria and chloroplasts. In addition, five genes coding for proteins homologous to FtsH (FtsHi 1-5) have been detected in the genome, lacking the conserved zinc-binding motif HEXXH, which presumably renders them inactive for proteolysis. These inactive FtsHs as well as nine of the active FtsHs are thought to be localized in the chloroplast. In this article, we shortly summarize the recent findings on plastidic FtsH proteases in text and figures. We will mainly focus on FtsH 1, 2, 5 and 8 localized in the thylakoid membrane and known for their importance in photosynthesis.

The Structural and Functional Basis of the p97/Valosin-containing Protein (VCP)-interacting Motif (VIM): MUTUALLY EXCLUSIVE BINDING OF COFACTORS TO THE N-TERMINAL DOMAIN OF p97

The AAA (ATPase associated with various cellular activities) ATPase p97, also referred to as valosin-containing protein (VCP), mediates essential cellular processes, including ubiquitin-dependent protein degradation, and has been linked to several human proteinopathies. p97 interacts with multiple cofactors via its N-terminal (p97N) domain, a subset of which contain the VCP-interacting motif (VIM). We have determined the crystal structure of the p97N domain in complex with the VIM of the ubiquitin E3 ligase gp78 at 1.8 Å resolution. The α-helical VIM peptide binds into a groove located in between the two subdomains of the p97N domain. Interaction studies of several VIM proteins reveal that these cofactors display dramatically different affinities, ranging from high affinity interactions characterized by dissociation constants of ∼20 nm for gp78 and ANKZF1 to only weak binding in our assays. The contribution of individual p97 residues to VIM binding was analyzed, revealing that identical substitutions do not affect all cofactors in the same way. Taken together, the biochemical and structural studies define the framework for recognition of VIM-containing cofactors by p97. Of particular interest to the regulation of p97 by its cofactors, our structure reveals that the bound α-helical peptides of VIM-containing cofactors overlap with the binding site for cofactors containing the ubiquitin regulatory X (UBX) domain present in the UBX protein family or the ubiquitin-like domain of NPL4 as further corroborated by biochemical data. These results extend the concept that competitive binding is a crucial determinant in p97-cofactor interactions.

Ubp15p, a Ubiquitin Hydrolase Associated with the Peroxisomal Export Machinery

Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.

A novel role for the AAA ATPase spastin as a HOXA10 transcriptional corepressor in Ishikawa endometrial cells.

Homeobox A10 (HOXA10), a transcription factor required for uterine development and embryo receptivity, functions downstream of estrogen and progesterone in uterine endometrium. HOXA10 represses endometrial expression of empty spiracles homeobox 2 (EMX2), the human ortholog of Drosophila empty spiracles. The ATPases associated with various cellular activities (AAA) ATPase spastin has a well-characterized role in neurotransmitter trafficking. In this study, we characterize a novel role of spastin in transcriptional regulation. We identified spastin as a novel component of the HOXA10 transcriptional complex in Ishikawa nuclear extracts by immunoprecipitation and mass spectrophotometry. Using EMX2 as a model endometrial HOXA10 target gene, we show that the HOXA10-spastin corepressor complex bound the EMX2 promoter in chromatin immunoprecipitation assays. HOXA10 has been previously shown to repress endometrial EMX2 expression. We further observed that, although cotransfection of HOXA10 and spastin continued to repress endometrial EMX2-luciferase expression, the repression was reversed when spastin small interfering RNA was cotransfected with HOXA10. Mutations in the nuclear localization signal sequences of spastin abrogated not only its nuclear translocation but also its colocalization with HOXA10 as well as reversed EMX2-luciferase repression. Here, we describe a novel role for the AAA ATPase spastin in Ishikawa cells as a HOXA10 corepressor of EMX2. Uterine EMX2 levels are inversely related to embryo implantation rates. HOXA10 acts downstream of progesterone and has been shown to facilitate embryo implantation through regulation of endometrial EMX2 expression. Endometrial spastin, therefore, likely has a novel function downstream of estrogen and progesterone in implantation biology as a cofactor of HOXA10.

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

The AAA (ATPase-associated with various cellular activities) ATPase p97 acts on diverse substrate proteins to partake in various cellular processes such as membrane fusion and endoplasmic reticulum-associated degradation (ERAD). In membrane fusion, p97 is thought to function in analogy to the related ATPase NSF (N-ethylmaleimide-sensitive fusion protein), which promotes membrane fusion by disassembling a SNARE complex. In ERAD, p97 dislocates misfolded proteins from the ER membrane to facilitate their turnover by the proteasome. Here, we identify a novel function of p97 in endocytic trafficking by establishing the early endosomal autoantigen 1 (EEA1) as a new p97 substrate. We demonstrate that a fraction of p97 is localized to the early endosome membrane, where it binds EEA1 via the N-terminal C2H2 zinc finger domain. Inhibition of p97 either by siRNA or a pharmacological inhibitor results in clustering and enlargement of early endosomes, which is associated with an altered trafficking pattern for an endocytic cargo. Mechanistically, we show that p97 inhibition causes increased EEA1 self-association at the endosome membrane. We propose that p97 may regulate the size of early endosomes by governing the oligomeric state of EEA1.

Cdc48 and Cofactors Npl4-Ufd1 Are Important for G1 Progression during Heat Stress by Maintaining Cell Wall Integrity in Saccharomyces cerevisiae

The ubiquitin-selective chaperone Cdc48, a member of the AAA (ATPase Associated with various cellular Activities) ATPase superfamily, is involved in many processes, including endoplasmic reticulum-associated degradation (ERAD), ubiquitin- and proteasome-mediated protein degradation, and mitosis. Although Cdc48 was originally isolated as a cell cycle mutant in the budding yeast Saccharomyces cerevisiae, its cell cycle functions have not been well appreciated. We found that temperature-sensitive cdc48-3 mutant is largely arrested at mitosis at 37°C, whereas the mutant is also delayed in G1 progression at 38.5°C. Reporter assays show that the promoter activity of G1 cyclin CLN1, but not CLN2, is reduced in cdc48-3 at 38.5°C. The cofactor npl4-1 and ufd1-2 mutants also exhibit G1 delay and reduced CLN1 promoter activity at 38.5°C, suggesting that Npl4-Ufd1 complex mediates the function of Cdc48 at G1. The G1 delay of cdc48-3 at 38.5°C is a consequence of cell wall defect that over-activates Mpk1, a MAPK family member important for cell wall integrity in response to stress conditions including heat shock. cdc48-3 is hypersensitive to cell wall perturbing agents and is synthetic-sick with mutations in the cell wall integrity signaling pathway. Our results suggest that the cell wall defect in cdc48-3 is exacerbated by heat shock, which sustains Mpk1 activity to block G1 progression. Thus, Cdc48-Npl4-Ufd1 is important for the maintenance of cell wall integrity in order for normal cell growth and division.