Cdc48/p97 segregase is modulated by cyclin‐dependent kinase to determine cyclin fate during G1 progression

Cells sense myriad signals during G1, and a rapid response to prevent cell cycle entry is of crucial importance for proper development and adaptation. Cln3, the most upstream G1 cyclin in budding yeast, is an extremely short‐lived protein subject to ubiquitination and proteasomal degradation. On the other hand, nuclear accumulation of Cln3 depends on chaperones that are also important for its degradation. However, how these processes are intertwined to control G1‐cyclin fate is not well understood. Here, we show that Cln3 undergoes a challenging ubiquitination step required for both degradation and full activation. Segregase Cdc48/p97 prevents degradation of ubiquitinated Cln3, and concurrently stimulates its ER release and nuclear accumulation to trigger Start. Cdc48/p97 phosphorylation at conserved Cdk‐target sites is important for recruitment of specific cofactors and, in both yeast and mammalian cells, to attain proper G1‐cyclin levels and activity. Cdk‐dependent modulation of Cdc48 would subjugate G1 cyclins to fast and reversible state switching, thus arresting cells promptly in G1 at developmental or environmental checkpoints, but also resuming G1 progression immediately after proliferative signals reappear.     

An ALS disease mutation in Cdc48/p97 impairs 20S proteasome binding and proteolytic communication

Cdc48 (also known as p97 or VCP) is an essential and highly abundant, double-ring AAA+ ATPase, which is ubiquitous in archaea and eukaryotes. In archaea, Cdc48 ring hexamers play a direct role in quality control by unfolding and translocating protein substrates into the degradation chamber of the 20S proteasome. Whether Cdc48 and 20S cooperate directly in protein degradation in eukaryotic cells is unclear. Two regions of Cdc48 are important for 20S binding, the pore-2 loop at the bottom of the D2 AAA+ ring and a C-terminal tripeptide. Here, we identify an aspartic acid in the pore-2 loop as an important element in 20S recognition. Importantly, mutation of this aspartate in human Cdc48 has been linked to familial amyotrophic lateral sclerosis (ALS). In archaeal or human Cdc48 variants, we find that mutation of this pore-2 residue impairs 20S binding and proteolytic communication but does not affect the stability of the hexamer or rates of ATP hydrolysis and protein unfolding. These results suggest that human Cdc48 interacts functionally with the 20S proteasome.     

A Conserved Protein with AN1 Zinc Finger and Ubiquitin-like Domains Modulates Cdc48 (p97) Function in the Ubiquitin-Proteasome Pathway

Regulated protein degradation mediated by the ubiquitin-proteasome system (UPS) is critical to eukaryotic protein homeostasis. Often vital to degradation of protein substrates is their disassembly, unfolding, or extraction from membranes. These processes are catalyzed by the conserved AAA-ATPase Cdc48 (also known as p97). Here we characterize the Cuz1 protein (Cdc48-associated UBL/zinc finger protein-1), encoded by a previously uncharacterized arsenite-inducible gene in budding yeast. Cuz1, like its human ortholog ZFAND1, has both an AN1-type zinc finger (Zf_AN1) and a divergent ubiquitin-like domain (UBL). We show that Cuz1 modulates Cdc48 function in the UPS. The two proteins directly interact, and the Cuz1 UBL, but not Zf_AN1, is necessary for binding to the Cdc48 N-terminal domain. Cuz1 also associates, albeit more weakly, with the proteasome, and the UBL is dispensable for this interaction. Cuz1-proteasome interaction is strongly enhanced by exposure of cells to the environmental toxin arsenite, and in a proteasome mutant, loss of Cuz1 enhances arsenite sensitivity. Whereas loss of Cuz1 alone causes only minor UPS degradation defects, its combination with mutations in the Cdc48^Npl4-Ufd1 complex leads to much greater impairment. Cuz1 helps limit the accumulation of ubiquitin conjugates on both the proteasome and Cdc48, suggesting a possible role in the transfer of ubiquitylated substrates from Cdc48 to the proteasome or in their release from these complexes.     

Insights into the regulation of neuronal viability by nucleophosmin/B23

The vastness of the neuronal network that constitutes the human brain proves challenging when trying to understand its complexity. Furthermore, due to the senescent state they enter into upon maturation, neurons lack the ability to regenerate in the face of insult, injury or death. Consequently, their excessive death can be detrimental to the proper functioning of the brain. Therefore, elucidating the mechanisms regulating neuronal survival is, while challenging, of great importance as the incidence of neurological disease is becoming more prevalent in today’s society. Nucleophosmin/B23 (NPM) is an abundant and ubiquitously expressed protein that regulates vital cellular processes such as ribosome biogenesis, cell proliferation and genomic stability. As a result, it is necessary for proper embryonic development, but has also been implicated in many cancers. While highly studied in the context of proliferative cells, there is a lack of understanding NPM’s role in post-mitotic neurons. By exploring its role in healthy neurons as well as its function in the regulation of cell death and neurodegeneration, there can be a better understanding of how these diseases initiate and progress. Owing to what is thus far known about its function in the cell, NPM could be an attractive therapeutic target in the treatment of neurodegenerative diseases.     

A longevity protein,Lag2, interacts with SCF complex and regulates SCF function

SCF‐type E3‐ubiquitin ligases control numerous cellular processes through the ubiquitin‐proteasome pathway. However, the regulation of SCF function remains largely uncharacterized. Here, we report a novel SCF complex‐interacting protein, Lag2, in Saccharomyces cerevisiae. Lag2 interacts with the SCF complex under physiological conditions. Lag2 negatively controls the ubiquitylation activities of SCF E3 ligase by interrupting the association of Cdc34 to SCF complex. Overexpression of Lag2 increases unrubylated Cdc53, whereas deletion of lag2, together with the deletions of dcn1 and jab1, results in the accumulation of Rub1‐modified Cdc53. In vitro rubylation assays show that Lag2 inhibits the conjugation of Rub1 to Cdc53 in competition with Dcn1, which suggest that Lag2 down‐regulates the rubylation of Cdc53 rather than promoting derubylation. Furthermore, Dcn1 hinders the association of Lag2 to Cdc53 in vivo. Finally, the deletion of lag2 combined with the deletion of either dcn1 or rub1 suppresses the growth of yeast cells. These observations thus indicate that Lag2 has a significant function in regulating the SCF complex by controlling its ubiquitin ligase activities and its rubylation cycle.     

Mechano-chemical effects of Ca(2+) in cross-linked troponin-C films

The ATPase Cdc48 is required for membrane fusion and protein degradation. Recently it has been suggested that Cdc48 in a complex with Ufd1 and Npl4 acts as an ubiquitin-dependent chaperone. Here it is shown that recombinant Cdc48 alone can distinguish between the native and the non-native conformation of model substrates. First, Cdc48 prevents luciferase from aggregating following a heat shock. Second, it inhibits the aggregation of rhodanese upon dilution. Third, Cdc48 binds specifically to heat-denatured luciferase. These chaperone-like functions seem to be independent of ATPase activity. Furthermore, Cdc48 can act as a co-chaperone in the Hsc70–Hsp40 chaperone system. These results show that Cdc48 possesses chaperone-like activities and can functionally interact with Hsc70. Cdc48’s ability to recognise denatured proteins can also be a source of unspecific binding in biochemical interaction experiments.     

Identification of ubiquilin, a novel presenilin interactor that increases presenilin protein accumulation

Mutations in the highly homologous presenilin genes encoding presenilin-1 and presenilin-2 (PS1 and PS2) are linked to early-onset Alzheimer's disease (AD). However, apart from a role in early development, neither the normal function of the presenilins nor the mechanisms by which mutant proteins cause AD are well understood. We describe here the properties of a novel human interactor of the presenilins named ubiquilin. Yeast two-hybrid (Y2H) interaction, glutathione S-transferase pull-down experiments, and colocalization of the proteins expressed in vivo, together with coimmunoprecipitation and cell fractionation studies, provide compelling evidence that ubiquilin interacts with both PS1 and PS2. Ubiquilin is noteworthy since it contains multiple ubiquitin-related domains typically thought to be involved in targeting proteins for degradation. However, we show that ubiquilin promotes presenilin protein accumulation. Pulse-labeling experiments indicate that ubiquilin facilitates increased presenilin synthesis without substantially changing presenilin protein half-life. Immunohistochemistry of human brain tissue with ubiquilin-specific antibodies revealed prominent staining of neurons. Moreover, the anti-ubiquilin antibodies robustly stained neurofibrillary tangles and Lewy bodies in AD and Parkinson's disease affected brains, respectively. Our results indicate that ubiquilin may be an important modulator of presenilin protein accumulation and that ubiquilin protein is associated with neuropathological neurofibrillary tangles and Lewy body inclusions in diseased brain.     

Protein dislocation from the endoplasmic reticulum--pulling out the suspect

Proteins that fail to fold properly as well as constitutive or regulated short-lived proteins of the endoplasmic reticulum are subjected to proteolysis by cytosolic 26S proteasomes. This process is known as endoplasmic reticulum-associated protein degradation. In order to become accessible to the proteasome of this system substrates must first be retrogradely transported from the endoplasmic reticulum into the cytosol, in a process termed dislocation. This export step seems to be accompanied by polyubiquitination of such molecules. Surprisingly, protein dislocation from the endoplasmic reticulum seems to require at least some components that mediate import into this compartment. However, protein import and export display differences in the mechanism that provides the driving force and ensures directionality. Of special interest is the cytoplasmic Cdc48p/Npl4p/Ufd1p complex, which is required for the degradation of various endoplasmic reticulum-associated protein degradation substrates and seems to function in a step after polyubiquitination but before proteasomal digestion. In this review, we will summarize our knowledge on protein export during endoplasmic reticulum-associated protein degradation and discuss the possible function of certain components involved in this process.     

Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects.     

The evolutionary context of robust and redundant cell biological mechanisms

The robustness of biological processes to perturbations has so far been mainly explored in unicellular organisms; multicellular organisms have been studied for developmental processes or in the special case of redundancy between gene duplicates. Here we explore the robustness of cell biological mechanisms of multicellular organisms in an evolutionary context. We propose that the reuse of similar cell biological mechanisms in different cell types of the same organism has evolutionary implications: (1) the maintenance of apparently redundant mechanisms over evolutionary time may in part be explained by their differential requirement in various cell types; (2) the relative requirement for two alternative mechanisms may evolve among homologous cells in different organisms. We present examples of cell biological processes, such as centrosome separation in prophase, spindle formation or cleavage furrow positioning, that support the first proposition. We propose experimental tests of these hypotheses.     

Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism

Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin''s in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.     

The small GTPase Arf1 modulates mitochondrial morphology and function

The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER–mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.     

Antibodies and protein misfolding: From structural research tools to therapeutic strategies

Protein misfolding disorders, including the neurodegenerative conditions Alzheimer's disease (AD) and Parkinson's disease (PD) represent one of the major medical challenges or our time. The underlying molecular mechanisms that govern protein misfolding and its links with disease are very complex processes, involving the formation of transiently populated but highly toxic molecular species within the crowded environment of the cell and tissue. Nevertheless, much progress has been made in understanding these events in recent years through innovative experiments and therapeutic strategies, and in this review we present an overview of the key roles of antibodies and antibody fragments in these endeavors. We discuss in particular how these species are being used in combination with a variety of powerful biochemical and biophysical methodologies, including a range of spectroscopic and microscopic techniques applied not just in vitro but also in situ and in vivo, both to gain a better understanding of the mechanistic nature of protein misfolding and aggregation and also to design novel therapeutic strategies to combat the family of diseases with which they are associated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains

A member of the family of ATPases associated with diverse cellular activities, called p97 in mammals and Cdc48 in yeast, associates with the cofactor Ufd1-Npl4 to move polyubiquitinated polypeptides from the endoplasmic reticulum (ER) membrane into the cytosol for their subsequent degradation by the proteasome. Here, we have studied the mechanism by which the p97-Ufd1-Npl4 complex functions in this retrotranslocation pathway. Substrate binding occurs when the first ATPase domain of p97 (D1 domain) is in its nucleotide-bound state, an interaction that also requires an association of p97 with the membrane through its NH2-terminal domain. The two ATPase domains (D1 and D2) of p97 appear to alternate in ATP hydrolysis, which is essential for the movement of polypeptides from the ER membrane into the cytosol. The ATPase itself can interact with nonmodified polypeptide substrates as they emerge from the ER membrane. Polyubiquitin chains linked by lysine 48 are recognized in a synergistic manner by both p97 and an evolutionarily conserved ubiquitin-binding site at the NH2 terminus of Ufd1. We propose a dual recognition model in which the ATPase complex binds both a nonmodified segment of the substrate and the attached polyubiquitin chain; polyubiquitin binding may activate the ATPase p97 to pull the polypeptide substrate out of the membrane.     

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.     

VCIP135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic Golgi fragments

The AAA-ATPase p97/Cdc48 functions in different cellular pathways using distinct sets of adapters and other cofactors. Together with its adaptor Ufd1-Npl4, it extracts ubiquitylated substrates from the membrane for subsequent delivery to the proteasome during ER-associated degradation. Together with its adaptor p47, on the other hand, it regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis. The finding of a ubiquitin-binding domain in p47 raises the question as to whether the ubiquitin-proteasome system is also involved in membrane fusion events. Here, we show that p97-p47-mediated reassembly of Golgi cisternae requires ubiquitin, but is not dependent on proteasome-mediated proteolysis. Instead, it requires the deubiquitinating activity of one of its cofactors, VCIP135, which reverses a ubiquitylation event that occurs during mitotic disassembly. Together, these data reveal a cycle of ubiquitylation and deubiquitination that regulates Golgi membrane dynamics during mitosis. Furthermore, they represent the first evidence for a proteasome-independent function of p97/Cdc48.     

Ubiquitin receptors and ERAD: a network of pathways to the proteasome

The elimination of misfolded proteins, known as protein quality control, is an essential cellular process. Removal of misfolded proteins from the secretory pathway depends on their recognition in the endoplasmic reticulum (ER) followed by their retrograde transport into the cytosol for degradation. The AAA-ATPase Cdc48/p97 facilitates the translocation of misfolded ER-proteins into the cytosol. Cdc48/p97 can dock onto the ER-membrane via direct interaction with ER-membrane proteins and/or indirectly via its substrate-recruiting cofactors, which interact with the ubiquitylated substrates at the membrane. This tight interaction in conjunction with the conformational changes induced upon ATP hydrolysis within Cdc48/p97 is thought to provide the driving force for the translocation reaction. Subsequently, a series of protein-protein interactions between the Cdc48/p97 complex, its cofactors, and the ubiquitylated substrates is instrumental for the proper delivery of the ER substrates to the proteasome. These protein-protein interactions are governed mainly by ubiquitin-fold and ubiquitin-binding domains.