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Membrane Traffic
... VCP is a highly conserved, ubiquitously and abundantly expressed ATPase. Indeed, VCP may make up as much as 1% of all cytoplasmic protein [1]. VCP mediates ubiquitindependent cellular processes through the ubiquitin-proteasome system (UPS) [2], protein quality control [3,4], transcription factor processing [5], membrane fusion [6], cell cycle control [7], and regulation of autophagy [8,9]. ...
In this work, we review clinical features and genetic diagnosis of diseases caused by mutations in the gene encoding valosin-containing protein (VCP/p97), the functionally diverse AAA-ATPase. VCP is crucial to a multitude of cellular functions including protein quality control, stress granule formation and clearance, and genomic integrity functions, among others. Pathogenic mutations in VCP cause multisystem proteinopathy (VCP-MSP), an autosomal dominant, adult-onset disorder causing dysfunction in several tissue types. It can result in complex neurodegenerative conditions including inclusion body myopathy, frontotemporal dementia, amyotrophic lateral sclerosis, or combinations of these. There is also an association with other neurodegenerative phenotypes such as Alzheimer-type dementia and Parkinsonism. Non-neurological presentations include Paget disease of bone and may also include cardiac dysfunction. We provide a detailed discussion of genotype-phenotype correlations, recommendations for genetic diagnosis, and genetic counselling implications of VCP-MSP.
Key message
We demonstrate that the C-terminus of OsCDC48 is essential for maintaining its full ATPase activity and OsCDC48/48E interaction is required to modulate cellular processes and plant survival in rice.
Abstract
Cell division cycle 48 (CDC48) belongs to the superfamily protein of ATPases associated with diverse cellular activities (AAA). We previously isolated a rice CDC48 mutant (psd128) displaying premature senescence and death phenotype. Here, we showed that OsCDC48 (Os03g0151800) interacted with OsCDC48E (Os10g0442600), a homologue of OsCDC48, to control plant survival in rice. OsCDC48E knockout plants exhibited similar behavior to psd128 with premature senescence and plant death. Removal of the C-terminus of OsCDC48 caused altered expression of cell cycle-related genes, changed the percentage of cells in G1 and G2/M phases, and abolished the interaction between OsCDC48 itself and between OsCDC48 and OsCDC48E, respectively. Furthermore, the truncated OsCDC48–PSD128 protein lacking the C-terminal 27 amino acid residues showed a decreased level of ATPase activity. Overexpression of OsCDC48–psd128 resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, accumulation of reactive oxygen species and decreased plant tiller numbers while overexpression of OsCDC48 also resulted in differential expression of AAA-ATPase associated genes leading to increased total ATPase activity, but increased plant tiller numbers and grain yield, indicating its potential utilization for yield improvement. Our results demonstrated that the C-terminal region of OsCDC48 was essential for maintaining the full ATPase activity and OsCDC48/48E complex might function in form of heteromultimers to modulate cellular processes and plant survival in rice.
Neuronal preconditioning in vitro or in vivo with a stressful but non-lethal stimulus leads to new protein expression that mediates a profound neuroprotection against glutamate excitotoxicity and experimental stroke. The proteins that mediate neuroprotection are relatively unknown and under discovery. Here we find that the expression of the AAA + ATPase Thorase is induced by preconditioning stimulation both in vitro and in vivo. Thorase provides neuroprotection in an ATP-dependent manner against oxygen–glucose deprivation (OGD) neurotoxicity or glutamate N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity in vitro. Knock-down of Thorase prevents the establishment of preconditioning induced neuroprotection against OGD or NMDA neurotoxicity. Transgenic overexpression of Thorase provides neuroprotection in vivo against middle cerebral artery occlusion (MCAO)-induced stroke in mice, while genetic deletion of Thorase results in increased injury in vivo following stroke. These results define Thorase as a neuroprotective protein and understanding Thorase signaling could offer a new therapeutic strategy for the treatment of neurologic disorders.
Lysosomal myopathies are hereditary myopathies characterized morphologically by the presence of autophagic vacuoles. In mammals, autophagy plays an important role in the turnover of cell components, particularly in response to starvation or glucagons. In normal muscle, autolysosomes or autophagosomes are typically inconspicuous. In distinct neuromuscular disorders, however, lysosomes become structurally abnormal and functionally impaired, leading to the accumulation of autophagic vacuoles in myofibers. In some instances, the accumulation of autophagic vacuoles can be a prominent feature, implicating autophagy as a contributor to disease pathomechanism and/or progression. At present, there are two disorders in the muscle which are associated with a primary defect in lysosomal proteins: Danon and Pompe disease. There are, in addition, nonlysosomal disorders that influence the mechanism of autophagy and present with accumulation of autophagic vacuoles in the muscle fibers. This chapter will be a brief discussion of these disorders (except for Pompe disease which will be discussed in Chapter 28) highlighting the role of autophagy in disease progression.
Cell enlargement is a necessary requisite for sustained growth of cells of all higher organisms. Close coupling of plasma membrane electron transport and growth was indicated from the beginning with the earliest investigations. The mechanistic basis for the correlation became apparent with the discovery of a protein disulfide–thiol interchange activity associated with the ENOX proteins. Rates of cell enlargement and rates of ENOX1 and ENOX2 activities in response to activators, inhibitors, anticancer drugs or overexpression are highly correlated. Additionally, rates of cell enlargement are periodic with major period lengths paralleling those of the ENOX proteins present. Both cell enlargement and ENOX activity are blocked by thiol reagents. Furthermore, cell enlargement is restricted to the protein disulfide–thiol interchange part of the ENOX cycle that alternates with the oxidative (NADH or hydroquinone) portion of the cycle. Recombinant ENOX proteins when incorporated into synthetic lipid vesicles with other required constituents (AAA-ATPase, ATP, defined thiol-rich protein source) recapitulate cell enlargement in a completely cell-free system. Implicit in these findings is that the mechanism of cell enlargement in both plants and animals is similar and an active process not obligatorily driven by biosynthesis or turgor. An energy requirement is universal and met at the cell surface through coupling with a plasma membrane p97 AAA-ATPase (ATPase Associated with Diverse Cellular Activities).
Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle.
Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification.
The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells.
This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents.
This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
Copyright © 2015. Published by Elsevier B.V.
Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.
Cdc48 (also called VCP and p97) is an abundant protein that plays essential regulatory functions in a broad array of cellular processes. Working with various cofactors, Cdc48 utilizes its ATPase activity to promote the assembly and disassembly of protein complexes. Here, we review key biological functions and regulation of Cdc48 in ubiquitin-related events. Given the broad employment of Cdc48 in cell biology and its intimate ties to human diseases (e.g., amyotrophic lateral sclerosis), studies of Cdc48 will bring significant insights into the mechanism and function of ubiquitin in health and diseases.
DNA-dependent protein kinase (DNA-PK) has an important role in the repair of DNA damage and regulates the radiation sensitivity of glioblastoma cells. The VCP (valosine-containing protein), a chaperone protein that regulates ubiquitin-dependent protein degradation, is phosphorylated by DNA-PK and recruited to DNA double-strand break sites to regulate DNA damage repair. However, it is not clear whether VCP is involved in DNA-PKcs (DNA-PK catalytic subunit) degradation or whether it regulates the radiosensitivity of glioblastoma. Our data demonstrated that DNA-PKcs was ubiquitinated and bound to VCP. VCP knockdown resulted in the accumulation of the DNA-PKcs protein in glioblastoma cells, and the proteasome inhibitor MG132 synergised this increase. As expected, this increase promoted the efficiency of DNA repair in several glioblastoma cell lines; in turn, this enhanced activity decreased the radiation sensitivity and prolonged the survival fraction of glioblastoma cells in vitro. Moreover, the VCP knockdown in glioblastoma cells reduced the survival time of the xenografted mice with radiation treatment relative to the control xenografted glioblastoma mice. In addition, the VCP protein was also downregulated in ∼25% of GBM tissues from patients (WHO, grade IV astrocytoma), and the VCP protein level was correlated with patient survival (R(2)=0.5222, P<0.05). These findings demonstrated that VCP regulates DNA-PKcs degradation and increases the sensitivity of GBM cells to radiation.
Alterations in the ubiquitin-proteasome system (UPS) have been reported in several neurodegenerative disorders characterized by protein misfolding and aggregation, including the polylgutamine diseases. Machado-Joseph disease (MJD) or Spinocerebellar Ataxia type 3 is caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene, which encodes a 42 kDa deubiquitinating enzyme (DUB), ataxin-3. We investigated ataxin-3 deubiquitinating activity and the functional relevance of ataxin-3 interactions with two proteins previously described to interact with ataxin-3, hHR23A and valosin-containing protein (VCP/p97). We confirmed ataxin-3 affinity for both hHR23A and VCP/p97. hHR23A and ataxin-3 were shown to co-localize in discrete nuclear foci, while VCP/p97 was primarily cytoplasmic. hHR23A and VCP/p97 recombinant proteins were added, separately or together, to normal and expanded ataxin-3 in in vitro deubiquitination assays to evaluate their influence on ataxin-3 activity. VCP/p97 was shown to be an activator specifically of wild-type ataxin-3, exhibiting no effect on expanded ataxin-3, In contrast, we observed no significant alterations in ataxin-3 enzyme kinetics or substrate preference in the presence of hHR23A alone or in combination with VCP. Based on our results we propose a model where ataxin-3 normally functions with its interactors to specify the cellular fate of ubiquitinated proteins.
Cdc48p/p97 is a cytosolic essential AAA chaperone, which regulates multiple cellular reactions in a ubiquitin-dependent manner. We have recently shown that Cdc48p exhibits positively cooperative ATPase activity and loss of the positive cooperativity results in yeast cell death. Here we show that loss of the positive cooperativity of the yeast Cdc48p ATPase activity led to severe mitochondrial aggregation. The actin cytoskeleton and distribution of the ER-mitochondria tethering complex (ERMES) were eliminated from the cause of the mitochondrial aggregation. Instead, a mitochondrial outer membrane protein Fzo1p, which is required for mitochondrial fusion, and components of ERMES, which is involved in mitochondrial morphology, were remarkably stabilized in the Cdc48p mutants. In the last couple of years, it was shown that Vms1p functions as a cofactor of Cdc48p for the function of protein degradation of mitochondrial outer membrane proteins. Nevertheless, we found that Vms1p was not involved in the Cdc48p-dependent mitochondrial aggregation and loss of Vms1p did not significantly affect degradation rates of proteins anchored to the mitochondrial outer membrane. These results suggest that Cdc48p controls mitochondrial morphology by regulating turnover of proteins involved in mitochondrial morphology in a Vms1p-independent manner.
CDC48/p97 is a conserved homohexameric AAA-ATPase chaperone required for a variety of cellular processes but whose role in the development of a multicellular model system has not been examined. Here, we have used reverse genetics, visualization of a functional Arabidopsis (Arabidopsis thaliana) CDC48 fluorescent fusion protein, and morphological analysis to examine the subcellular distribution and requirements for AtCDC48A in planta. Homozygous Atcdc48A T-DNA insertion mutants arrest during seedling development, exhibiting decreased cell expansion and displaying pleiotropic defects in pollen and embryo development. Atcdc48A insertion alleles show significantly reduced male transmission efficiency due to defects in pollen tube growth. Yellow fluorescent protein-AtCDC48A, a fusion protein that functionally complements the insertion mutant defects, localizes in the nucleus and cytoplasm and is recruited to the division mid-zone during cytokinesis. The pattern of nuclear localization differs according to the stage of the cell cycle and differentiation state. Inducible expression of an Atcdc48A Walker A ATPase mutant in planta results in cytokinesis abnormalities, aberrant cell divisions, and root trichoblast differentiation defects apparent in excessive root hair emergence. At the biochemical level, our data suggest that the endogenous steady-state protein level of AtCDC48A is dependent upon the presence of ATPase-active AtCDC48A. These results demonstrate that CDC48A/p97 is critical for cytokinesis, cell expansion, and differentiation in plants.
Valosin-containing protein (VCP) has been shown to colocalize with abnormal protein aggregates, such as nuclear inclusions of Huntington disease and Machado-Joseph disease, Lewy bodies in Parkinson disease. Several mis-sense mutations in the human VCP gene have been identified in patients suffering inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). Recently, we have shown that VCP possesses both aggregate-forming and aggregate-clearing activities. Here, we showed that in cells treated with proteasome inhibitors VCP first appeared as several small aggregates throughout the cells; and then, these small aggregates gathered together into a single big aggregate. Subcellular localization and ATPase activity of VCP clearly influenced the localization of the aggregates. Furthermore, all tested IBMPFD-causing mutant VCPs, possessed elevated ATPase activities and enhanced aggregate-forming activities in cultured cells. In Drosophila, these mutants and VCP(T761E), a super active VCP, did not appear to spontaneously induce eye degeneration, but worsened the phenotype when co-expressed with polyglutamines. Unexpectedly, these VCPs did not apparently change sizes and the amounts of polyglutamine aggregates in Drosophila eyes. Elevated ATPase activities, thus, may be a hidden primary defect causing IBMPFD pathological phenotypes, which would be revealed when abnormal proteins are accumulated, as typically observed in aging.
Inclusion body myopathy associated with Paget's disease of the bone and fronto-temporal dementia (IBMPFD) is a progressive
autosomal dominant disorder caused by mutations in p97/VCP (valosin-containing protein). p97/VCP is a member of the AAA+ (ATPase
associated with a variety of activities) protein family and participates in multiple cellular processes. One particularly
important role for p97/VCP is facilitating intracellular protein degradation. p97/VCP has traditionally been thought to mediate
the ubiquitin-proteasome degradation of proteins; however, recent studies challenge this dogma. p97/VCP clearly participates
in the degradation of aggregate-prone proteins, a process principally mediated by autophagy. In addition, IBMPFD mutations
in p97/VCP lead to accumulation of autophagic structures in patient and transgenic animal tissue. This is likely due to a
defect in p97/VCP-mediated autophagosome maturation. The following review will discuss the evidence for p97/VCP in autophagy
and how a disruption in this process contributes to IBMPFD pathogenesis.
p97/valosin-containing protein (VCP) is a member of the AAA family proteins, which plays various important roles in cells by using its ATPase activity. But mechanism of regulating its ATPase activity is mostly unknown. We report here that VCP is highly modified throughout the protein via acetylation and phosphorylation. In addition to six previously identified phosphorylation sites, we identified at least 14 serines, 14 threonines, 6 tyrosines and 22 lysines as potential modification sites. Interestingly, these sites included Lys251 and Lys524, which are very critical for the ATP binding in Walker A motif of D1 and D2 domains, respectively. It is notable that 16 sites are in the N-terminal region and 16 sites are clustered in D2alpha domain (from Pro646 to Gly765). Indeed, amino acid substitution of Lys696 and Thr761 profoundly affect VCP ATPase activities. From these results, we propose that D2alpha domain acts as a VCP ATPase Regulatory domain or "VAR domain". VCP modifications including those in this VAR domain may endorse adaptive and multiple functions to VCP in different cell conditions such as in the cell cycle and with abnormal protein accumulation.
Bacterial conjugation is an example of macromolecular trafficking between cells and responsible for the spreading of antibiotic resistance among bacteria. It involves translocation of single-stranded DNA across membranes through a type IV secretion system. A coupling protein links the DNA-processing nucleoprotein complex, the relaxosome, with the transport apparatus during cell mating. In Escherichia coli plasmid R388 such a protein is TrwB, a basic integral inner-membrane nucleoside-triphosphate-binding protein. TrwB is the structural prototype for the type IV secretion system coupling proteins, a family of proteins essential for macromolecular transport between cells and export. The structure of a soluble TrwB variant unveils an elongated molecule with six equivalent protein units featuring a spherical quaternary structure, leaving a central channel. The structures of the non-liganded protein and four different complexes with substrate analogues and products allow the precise description of the active site architecture. The active sites are located at the interface between protomers, each of them shaped mainly by residues of one monomer, but including two crucial arginine residues belonging to the adjacent molecule. Upon substrate binding and putative hydrolysis, conformational changes are transferred from the external surface to the interior central channel.
The hexameric ATPase, N-ethylmaleimide-sensitive factor (NSF) is implicated in the release of neurotransmitters and in mediating fusion between intracellular membranes. Due to the conservation of proteins in constitutive and regulated membrane fusion reactions, NSF and its downstream targets have been predicted also to participate in fusion reactions underlying endocrine function, but there is little experimental evidence to support such a role for NSF in insect neuroendocrine secretion. Here we have characterized the NSF orthologue (MsNSF) from the endocrine model for development Manduca sexta. MsNSF is developmentally regulated in endocrine organs of the protocerebral complex. Enrichment of MsNSF in corpora cardiaca (CC) and not in corpora allata (CA) indicates that it might play a preferential role in releasing hormones produced in CC. Endocrine/paracrine cells of the enteric system in M. sexta exhibit selective MsNSF enrichment. Together the data point to a more selective participation of MsNSF in development of M. sexta by its involvement in a subset of factors, whereas other as-yet-unidentified homolog(s) might regulate secretion from CA and a large set of endocrine/paracrine cells. We further characterized the in vivo role of MsNSF by heterologous expression. In contrast to vertebrate NSF, MsNSF is functional in yeast membrane fusion in vivo. MsNSF rectifies defects in SEC18 (yeast NSF homologue) at nearly all discernible steps where Sec18p has been implicated in the biosynthetic route. This underscores the utility of our approach to delineate functional roles for proteins from systems that are not currently amenable to in vitro reconstitution.
Multivesicular endosomes (MVEs) are complex intracellular organelles that function in endocytosis. A major function of the endocytic pathway is to sort internalized macromolecules and membrane proteins. Appropriately sorted proteins, such as epidermal growth factor (EGF) receptor (EGFR), are incorporated into MVEs before transport to the lysosomal compartment, where degradation occurs. Thus, MVEs operate in the endosome-to-lysosome portion of the pathway. In yeast cells, where MVE formation has been extensively studied, the pathway terminates in the yeast vacuole, which is equivalent to the vertebrate lysosome. MVEs arise by invagination of the limiting membrane of an endosomal vesicle such that many small internal vesicles are formed, hence the term "multivesicular endosome." In part, the internalization and targeting of membrane proteins to the MVE involves ubiquitin, a small protein associated with protein degradation. In reticulocytes and certain antigen-presenting cells, MVEs are routed to the plasma membrane rather than the lysosome, releasing small vesicles called "exosomes" back into the extracellular space.
We utilize structurally targeted peptides to identify a "tC fusion switch" inherent to the coil domains of the neuronal t-SNARE that pairs with the cognate v-SNARE. The tC fusion switch is located in the membrane-proximal portion of the t-SNARE and controls the rate at which the helical bundle that forms the SNAREpin can zip up to drive bilayer fusion. When the fusion switch is "off" (the intrinsic state of the t-SNARE), zippering of the helices from their membrane-distal ends is impeded and fusion is slow. When the tC fusion switch is "on," fusion is much faster. The tC fusion switch can be thrown by a peptide that corresponds to the membrane-proximal half of the cognate v-SNARE, and binds reversibly to the cognate region of the t-SNARE. This structures the coil in the membrane-proximal domain of the t-SNARE and accelerates fusion, implying that the intrinsically unstable coil in that region is a natural impediment to the completion of zippering, and thus, fusion. Proteins that stabilize or destabilize one or the other state of the tC fusion switch would exert fine temporal control over the rate of fusion after SNAREs have already partly zippered up.
Abnormal protein accumulation and cell death with cytoplasmic vacuoles are hallmarks of several neurodegenerative disorders. We previously identified p97/valosin-containing protein (VCP), an AAA ATPase with two conserved ATPase domains (D1 and D2), as an interacting partner of the Machado-Joseph disease (MJD) protein with expanded polyglutamines that causes Machado-Joseph disease. To reveal its pathophysiological roles in neuronal cells, we focused on its ATPase activity. We constructed and characterized PC12 cells expressing wild-type p97/VCP and p97(K524A), a D2 domain mutant. The expression level, localization, and complex formation of both proteins were indistinguishable, but the ATPase activity of p97(K524A) was much lower than that of the wild type. p97(K524A) induced cytoplasmic vacuoles that stained with an endoplasmic reticulum (ER) marker, and accumulation of polyubiquitinated proteins in the nuclear and membrane but not cytoplasmic fractions was observed, together with the elevation of ER stress markers. These results show that p97/VCP is essential for degrading membrane-associated ubiquitinated proteins and that profound deficits in its ATPase activity severely affect ER quality control, leading to abnormal ER expansion and cell death. Excessive accumulation of misfolded proteins may inactivate p97/VCP in several neurodegenerative disorders, eventually leading to the neurodegenerations.
Three protein motors have been unambiguously identified as rotary engines: the bacterial flagellar motor and the two motors that constitute ATP synthase (F(0)F(1) ATPase). Of these, the bacterial flagellar motor and F(0) motors derive their energy from a transmembrane ion-motive force, whereas the F(1) motor is driven by ATP hydrolysis. Here, we review the current understanding of how these protein motors convert their energy supply into a rotary torque.
The closely related proteins prohibitin (p32) and prohibitone (p37) are evolutionarily conserved with homologues found from cyanobacteria to man. They are thought to be exclusively mitochondrial and have been assigned many-rather different-functions, ranging from a role in lifespan, in mitochondrial inheritance and as chaperones of mitochondrial proteases in yeast. Evidence for a localisation outside of mitochondria has been brought forward in mammalian cells, where they influence cell-cycle progression and are found in association with cell surface receptors. We have employed a yeast two-hybrid screen to identify other interacting proteins and have identified alpha-actinin and annexin A2 as binding partners for prohibitin and prohibitone. Coprecipitation experiments supported the putative binding between prohibitin and prohibitone on the one hand and annexin A2 or alpha-actinin on the other hand in intact cells. Surface plasmon resonance analysis was used to determine relative affinities between prohibitin and alpha-actinin and between prohibitone and annexin A2 and alpha-actinin, respectively. We further show that prohibitin and prohibitone can also form homomeric (preferentially tetrameric) and heteromultimeric complexes, with significant affinities.
Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.
A topic that is keeping cell biologists across several fields occupied is how the AAA ATPase p97 can have so many apparently unrelated functions. A recent model that proposed sets of adaptors for p97 selected according to the type of p97 activity seemed to afford a simple solution. For example, one known adaptor, the Ufd1-Npl4 complex, has been implicated in ubiquitin-dependent proteolysis whereas another, p47, is an essential co-factor for membrane fusion. However, further investigation has revealed that the situation is more complicated. Both Ufd1-Npl4 and p47 adaptors bind ubiquitin, and so their activities may be more closely related than first thought. A role for ubiquitin in p97-dependent membrane fusion is a particularly surprising development with no obvious explanation. However, some clues may be found from looking at the role of ubiquitin and the AAA ATPase Vps4 during sorting on the endocytic pathway.
The endoplasmic reticulum (ER) regulates protein synthesis, protein folding and trafficking, cellular responses to stress and intracellular calcium (Ca(2+)) levels. Alterations in Ca(2+) homeostasis and accumulation of misfolded proteins in the ER cause ER stress that ultimately leads to apoptosis. Prolonged ER stress is linked to the pathogenesis of several different neurodegenerative disorders. Apoptosis is a form of cell death that involves the concerted action of a number of intracellular signaling pathways including members of the caspase family of cysteine proteases. The two main apoptotic pathways, the death receptor ('extrinsic') and mitochondrial ('intrinsic') pathways, are activated by caspase-8 and -9, respectively, both of which are found in the cytoplasm. Recent studies point to the ER as a third subcellular compartment implicated in apoptotic execution. Here, we review evidence for the contribution of various cellular molecules that contribute to ER stress and subsequent cellular death. It is hoped that dissection of the molecular components and pathways that alter ER structure and function and ultimately promote cellular death will provide a framework for understanding degenerative disorders that feature misfolded proteins.
Dorfin, a RING-IBR type ubiquitin ligase (E3), can ubiquitylate mutant superoxide dismutase 1, the causative gene of familial amyotrophic lateral sclerosis (ALS). Dorfin is located in ubiquitylated inclusions (UBIs) in various neurodegenerative disorders, such as ALS and Parkinson's disease (PD). Here we report that Valosin-containing protein (VCP) directly binds to Dorfin and that VCP ATPase activity profoundly contributes to the E3 activity of Dorfin. High through-put analysis using mass spectrometry identified VCP as a candidate of Dorfin-associated protein. Glycerol gradient centrifugation analysis showed that endogenous Dorfin consisted of a 400-600-kDa complex and was co-immunoprecipitated with endogenous VCP. In vitro experiments showed that Dorfin interacted directly with VCP through its C-terminal region. These two proteins were colocalized in aggresomes in HEK293 cells and UBIs in the affected neurons of ALS and PD. VCP(K524A), a dominant negative form of VCP, reduced the E3 activity of Dorfin against mutant superoxide dismutase 1, whereas it had no effect on the autoubiquitylation of Parkin. Our results indicate that VCPs functionally regulate Dorfin through direct interaction and that their functional interplay may be related to the process of UBI formation in neurodegenerative disorders, such as ALS or PD.
The type III protein secretion system (TTSS) is a complex organelle in the envelope of many Gram-negative bacteria; it delivers potentially hundreds of structurally diverse bacterial virulence proteins into plant and animal cells to modulate host cellular functions. Recent studies have revealed several basic features of this secretion system, including assembly of needle/pilus-like secretion structures, formation of putative translocation pores in the host membrane, recognition of N-terminal/5' mRNA-based secretion signals, and requirement of small chaperone proteins for optimal delivery and/or expression of effector proteins. Although most of our knowledge about the TTSS is derived from studies of mammalian pathogenic bacteria, similar and unique features are learned from studies of plant pathogenic bacteria. Here, we summarize the most salient aspects of the TTSS, with special emphasis on recent findings.
N-Ethyl-maleimide-sensitive factor (NSF) plays a critical role in the regulation of exocytosis. NSF regulates exocytosis by interacting with a complex containing soluble NSF attachment protein receptor (SNARE) molecules, hydrolyzing ATP, and disassembling the SNARE complex. We hypothesized that peptide inhibitors of NSF would decrease exocytosis. We now report the development of a novel set of peptides that block exocytosis by inhibiting NSF activity. These NSF inhibitors are fusion polypeptides composed of an 11 amino acid human immunodeficiency virus transactivating regulatory protein (TAT) domain fused to a 22 amino acid NSF domain. These TAT-NSF fusion polypeptides cross endothelial cell membranes, inhibit NSF hydrolysis of ATP, decrease NSF disassembly of SNARE molecules, and block exocytosis of von Willebrand factor. Control peptides have no effect on exocytosis. TAT-NSF inhibitors administered to mice prolong the bleeding time. Blood concentrations of these TAT-NSF peptides rapidly decrease within 5 min after injection and then remain constant from 10 to 60 min after injection. These TAT-NSF compounds may be useful in the treatment of a variety of diseases in which exocytosis plays a prominent role, including myocardial infarction, stroke, thrombosis, and autoimmune disorders.
The AAA-peroxins Pex1p and Pex6p play a critical role in peroxisome biogenesis but their precise function remains to be established. These two peroxins consist of three distinct regions (N, D1, D2), two of which (D1, D2) contain a conserved approximately 230 amino acid cassette, which is common to all ATPases associated with various cellular activities (AAA). Here we show that Pex1p and Pex6p from Saccharomyces cerevisiae do interact in vivo. We assigned their corresponding binding sites and elucidated the importance of ATP-binding and -hydrolysis of Pex1p and Pex6p for their interaction. We show that the interaction of Pex1p and Pex6p involves their first AAA-cassettes and demonstrate that ATP-binding but not ATP-hydrolysis in the second AAA-cassette (D2) of Pex1p is required for the Pex1p-Pex6p interaction. Furthermore, we could prove that the second AAA-cassettes (D2) of both Pex1p and Pex6p were essential for peroxisomal biogenesis and thus probably comprise the overall activity of the proteins.
Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.
The AAA(+) ATPase component of magnesium chelatase (ChlI) drives the insertion of Mg(2+) into protoporphyrin IX; this is the first step in chlorophyll biosynthesis. We describe the ATPase activity, nucleotide binding kinetics, and structural organization of the ChlI protein. A consistent reaction scheme arises from our detailed steady state description of the ATPase activity of the ChlI subunit and from transient kinetic analysis of nucleotide binding. We provide the first demonstration of metal ion binding to a specific subunit of any of the multimeric chelatases and characterize binding of Mg(2+) to the free and MgATP(2)(-) bound forms of ChlI. Transient kinetic studies with the fluorescent substrate analogue TNP-ATP show that there are two forms of monomeric enzyme, which have distinct magnesium binding properties. Additionally, we describe the self-association properties of the subunit and provide a structural analysis of the multimeric ring formed by this enzyme in the presence of nucleotide. This single particle analysis demonstrates that this species has a 7-fold rotational symmetry, which is in marked contrast to most members of the AAA(+) family that tend to form hexamers.
We herein isolated a peroxisome-deficient Chinese hamster ovary mutant, ZPEG252, import-defective of peroxisome targeting signal 1 (PTS1)- and PTS2-proteins at 37 degrees C. The impaired protein import was restored at 30 degrees C, indicating a temperature-sensitive phenotype, similar to that of cells derived from patients with milder peroxisome biogenesis disorders such as infantile Refsum disease. PEX1 expression complemented the mutant phenotype of ZPEG252. Reverse transcription-PCR analysis indicated one point mutation at nucleotide residue 1817 changing a codon (GGG) for Gly(606) to a codon (GAG) for Glu(606) in the sequence for the Walker A1 motif of the AAA cassettes. This novel mutant Pex1pG606E was severely affected in binding to Pex6p at 37 degrees C, but not at 30 degrees C. Pex1pG606E was localized to peroxisomes at 30 degrees C, whilst it was discernible in a cytosolic staining pattern at 37 degrees C. Together, our findings demonstrate that Walker A1 motif of Pex1p is essential for Pex1p-Pex6p interaction and Pex1p targeting to peroxisomes.
An AAA-ATPase (ATPases Associated with a Variety of Cellular Activities) localized to the plasma membrane of soybean (Glycine max) was isolated, partially sequenced and cloned (SBPM AAA-ATPase). The protein with an apparent monomer molecular mass of about 97 kDa was isolated using a combination of anion exchange, preparative SDS-PAGE, reverse phase HPLC, and ATP affinity chromatography. The cDNA for the full-length SBPM AAA-ATPase was cloned by screening an expression library using an antibody against the highly conserved Walker B AAA-ATP-binding motif. Northern blot analysis detected one transcript of approximately 2700 bp. The full-length cDNA sequence was that previously obtained (GenBank Database; U20213) encoding a protein with two copies of the conserved AAA-ATP-binding motif and regions of sequence homology with other AAA-ATPases. Electron microscopic preparations of the recombinant SBPM AAA-ATPase revealed hexamers typically formed by these proteins. The cloned and expressed protein was identical to the protein isolated from the soybean plasma membrane as confirmed using antisera raised to a non-conserved region of the derived protein sequence and by N-terminal sequencing of peptides derived from the isolated protein.
A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichiometric proportions into liposomes together with albumin as a source of protein thiols. Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl phosphatidylcholines. The lipids were dried to a film and reconstituted into vesicles by resuspension in buffer containing the recombinant proteins in equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these vesicles enlarged in an ATP-dependent manner based on light-scattering measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized, the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX, the recombinant AAA-ATPase, a source of protein thiols and ATP all were required. In control experiments, no ATP-dependent vesicle enlargement was observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of ATP was without any effect when only the single proteins were incorporated into the lipid vesicles. A model has been developed whereby the plasma membrane AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX protein, to membrane structural proteins. Binding of ATP and subsequent hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby both thinning the membrane and increasing the vesicle surface.
Alterations in Ca2+ homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) cause ER stress that ultimately leads
to programmed cell death. Recent studies have shown that ER stress triggers programmed cell death via an alternative intrinsic
pathway of apoptosis that, unlike the intrinsic pathway described previously, is independent of Apaf-1 and cytochrome c. In the present work, we have used a set of complementary approaches, including two-dimensional gel electrophoresis coupled
with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and nano-liquid chromatography-electrospray
ionization mass spectrometry with tandem mass spectrometry, RNA interference, co-immunoprecipitation, immunodepletion of candidate
proteins, and reconstitution studies, to identify mediators of the ER stress-induced cell death pathway. Our data identify
two molecules, valosin-containing protein and apoptosis-linked gene-2 (ALG-2), that appear to play a role in mediating ER
stress-induced cell death.
An in-frame 3 bp deletion in the torsinA gene resulting in the loss of a glutamate residue at position 302 or 303 (torsinA DeltaE) is the major cause for early-onset torsion dystonia (DYT1). In addition, an 18 bp deletion in the torsinA gene resulting in the loss of residues 323-328 (torsinA Delta323-8) has also been associated with dystonia. Here we report that torsinA DeltaE and torsinA Delta323-8 mutations cause neuronal cell-type-specific mislocalization of torsinA protein to the nuclear envelope without affecting torsinA oligomerization. Furthermore, both dystonia-associated mutations destabilize torsinA protein in dopaminergic cells. We find that wild-type torsinA protein is degraded primarily through the macroautophagy-lysosome pathway. In contrast, torsinA DeltaE and torsinA Delta323-8 mutant proteins are degraded by both the proteasome and macroautophagy-lysosome pathways. Our findings suggest that torsinA mutation-induced premature degradation may contribute to the pathogenesis of dystonia via a loss-of-function mechanism and underscore the importance of both the proteasome and macroautophagy in the clearance of dystonia-associated torsinA mutant proteins.
We have discovered a ring-shaped particle of 12.5 nm diameter, 14.5S and apparent molecular weight of approximately 570,000 that displays 6-fold radial symmetry and is composed of a single kind of an acidic (pI approximately 5.5) polypeptide of Mr 97,000 (p97). Using antibodies to this protein we have detected its occurrence in a wide range of cells and tissues of diverse species from frog to man, including highly specialized cells such as mammalian erythrocytes and spermatozoa. In Xenopus laevis oocytes, the particle is found in both isolated nuclei and in manually enucleated ooplasms, which corresponds to immunofluorescence staining dispersed over both nucleoplasm and cytoplasm. The particle has a N-ethylmaleimide (NEM)-inhibitable Mg2(+)-ATPase activity, and its amino acid sequence, as deduced from cDNA clones, displays considerable homology to the mammalian NEM-sensitive fusion protein (NSF) and yeast Sec18p believed to be essential for vesicle fusion in secretory processes, indicating that these three proteins belong to the same multigene family.
A library of random 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.
At least two distinct ATPases, NSF and p97, are known to be involved in the heterotypic fusion of transport vesicles with their target membranes and the homotypic fusion of membrane compartments. The NSF-mediated fusion pathway is the best characterized, many of the components having been identified and their functions analysed. In contrast, none of the accessory proteins for the p97-mediated fusion pathway has been identified. Now we have identified the first such component, a protein of relative molecular mass 47,000 (p47), which forms a tight, stoichiometric complex with cytosolic p97 (one trimer of p47 per hexamer of p97). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. As a homologue of p47 exists in budding yeast, this indicates that it might also be involved in other membrane fusion reactions catalysed by p97, such as karyogamy.
Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.
The crystal structure of the delta' subunit of the clamp-loader complex of E. coli DNA polymerase III has been determined. Three consecutive domains in the structure are arranged in a C-shaped architecture. The N-terminal domain contains a nonfunctional nucleotide binding site. The catalytic component of the clamp-loader complex is the gamma subunit, which is homologous to delta'. A sequence-structure alignment suggests that nucleotides bind to gamma at an interdomain interface within the inner surface of the "C." The alignment is extended to other clamp-loader complexes and to the RuvB family of DNA helicases, and suggests that each of these is assembled from C-shaped components that can open and close the jaws of the "C" in response to ATP binding and hydrolysis.
N-ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic ATPase required for many intracellular vesicle fusion reactions. NSF consists of an amino-terminal region that interacts with other components of the vesicle trafficking machinery, followed by two homologous ATP-binding cassettes, designated D1 and D2, that possess essential ATPase and hexamerization activities, respectively. The crystal structure of D2 bound to Mg2+-AMPPNP has been determined at 1.75 A resolution. The structure consists of a nucleotide-binding and a helical domain, and it is unexpectedly similar to the first two domains of the clamp-loading subunit delta' of E. coli DNA polymerase III. The structure suggests several regions responsible for coupling of ATP hydrolysis to structural changes in full-length NSF.
The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.
AAA ATPases(those associated with various cellular activities) play important roles in numerous cellular activities including proteolysis, protein folding, membrane trafficking, cytoskeletal regulation, organelle biogenesis, DNA replication, and intracellular motility. Recent structural and enzymatic studies are providing clues into the properties of the conserved AAA domain that defines this large protein superfamily. In many cases, AAA domains assemble into hexamic rings that are likely to change their shape during the ATPase cycle. This nucleotide-dependent conformational switch may apply tension to bound proteins and thereby allow AAA proteins to unfold polypeptides, dissociate protein-protein interactions, or generate unidirectional motion along a track. Thus, AAA proteins may represent a broad class of mechanoenzymes that have evolved unique ways of using a fundamentally similar conformational change in many different biological settings.
p97, an abundant hexameric ATPase of the AAA family, is involved in homotypic membrane fusion. It is thought to disassemble SNARE complexes formed during the process of membrane fusion. Here, we report two structures: a crystal structure of the N-terminal and D1 ATPase domains of murine p97 at 2.9 A resolution, and a cryoelectron microscopy structure of full-length rat p97 at 18 A resolution. Together, these structures show that the D1 and D2 hexamers pack in a tail-to-tail arrangement, and that the N domain is flexible. A comparison with NSF D2 (ATP complex) reveals possible conformational changes induced by ATP hydrolysis. Given the D1 and D2 packing arrangement, we propose a ratchet mechanism for p97 during its ATP hydrolysis cycle.
AAA ATPases play central roles in cellular activities. The ATPase p97, a prototype of this superfamily, participates in organelle membrane fusion. Cryoelectron microscopy and single-particle analysis revealed that a major conformational change of p97 during the ATPase cycle occurred upon nucleotide binding and not during hydrolysis as previously hypothesized. Furthermore, our study indicates that six p47 adaptor molecules bind to the periphery of the ring-shaped p97 hexamer. Taken together, these results provide a revised model of how this and possibly other AAA ATPases can translate nucleotide binding into conformational changes of associated binding partners.
A cell-free system that mimics the reassembly of Golgi stacks at the end of mitosis requires two ATPases, NSF and p97, to rebuild Golgi cisternae. Morphological studies now show that alpha-SNAP, a component of the NSF pathway, can inhibit the p97 pathway, whereas p47, a component of the p97 pathway, can inhibit the NSF pathway. Anti-syntaxin 5 antibodies and a soluble, recombinant syntaxin 5 inhibited both pathways, suggesting that this t-SNARE is a common component. Biochemical studies confirmed this, showing that p47 binds directly to syntaxin 5 and competes for binding with alpha-SNAP. p47 also mediates the binding of p97 to syntaxin 5 and so plays an analogous role to alpha-SNAP, which mediates the binding of NSF.
The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle, karyogamy, and the maintenance of an intact organellar compartment. We showed previously that this process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Here we show that Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. We propose that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events.
The highly conserved ATPase p97, a member of the AAA-ATPases, is found in a complex with its co-factor p47 in rat liver cytosol. Previously it had been shown that p97-mediated reassembly of Golgi cisternae from mitotic Golgi fragments requires p47 which mediates the binding of p97 to a Golgi t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment factor receptor), syntaxin 5. Here we show that it also suppresses the ATPase activity of p97 by up to 85% in a dose-dependent and saturable manner suggesting that it has other roles in the membrane fusion cycle.
Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes.
Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein. We have used electron tomography to obtain low-resolution (2-2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T. acidophilum complex lacking the putative substrate binding domain.
The degradation of cytoplasmic proteins is an ATP-dependent process. Substrates are targeted to a single soluble protease, the 26S proteasome, in eukaryotes and to a number of unrelated proteases in prokaryotes. A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU) in Escherichia coli. Its protease component HslV shares approximately 20% sequence similarity and a conserved fold with 20S proteasome beta-subunits. HslU is a member of the Hsp100 (Clp) family of ATPases. Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV-the first structure of a complete set of components of an ATP-dependent protease. HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components. Instead, there is conformational flexibility and domain motion in HslU and a localized order-disorder transition in HslV. Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states. They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein, the prototype of an AAA-ATPase. A third, mostly alpha-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases.
The fusion of vesicles with target membranes is controlled by a complex network of protein-protein and protein-lipid interactions. Recent structures of the SNARE complex, synaptotagmin III, nSec1, domains of NSF and its adaptor SNAP, along with Rab3 and some of its effectors, provide the framework for developing molecular models of vesicle fusion and for designing experiments to test these models. Ultimately, this knowledge of the structures of higher-order complexes and their dynamic behavior will allow us to obtain a full understanding of the vesicle fusion protein machinery.
HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.
- A T Brü Nger
Brü nger, A.T. (2000). Curr. Opin. Neurobiol. 10, 293-302.
- Vale R.D