The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast

The regulator of ATPase of vacuoles and endosomes (RAVE) complex is implicated in vacuolar H⁺-translocating ATPase (V-ATPase) assembly and activity. In yeast, rav1∆ mutants exhibit a Vma⁻ growth phenotype characteristic of loss of V-ATPase activity only at high temperature. Synthetic genetic analysis identified mutations that exhibit a full, temperature-independent Vma⁻ growth defect when combined with the rav1∆ mutation. These include class E vps mutations, which compromise endosomal sorting. The synthetic Vma⁻ growth defect could not be attributed to loss of vacuolar acidification in the double mutants, as there was no vacuolar acidification in the rav1∆ mutant. The yeast V-ATPase a subunit is present as two isoforms, Stv1p in Golgi and endosomes and Vph1p in vacuoles. Rav1p interacts directly with the N-terminal domain of Vph1p. STV1 overexpression suppressed the growth defects of both rav1∆ and rav1∆vph1∆, and allowed RAVE-independent assembly of active Stv1p-containing V-ATPases in vacuoles. Mutations causing synthetic genetic defects in combination with rav1∆ perturbed the normal localization of Stv1–green fluorescent protein. We propose that RAVE is necessary for assembly of Vph1-containing V-ATPase complexes but not Stv1-containing complexes. Synthetic Vma⁻ phenotypes arise from defects in Vph1p-containing complexes caused by rav1∆, combined with defects in Stv1p-containing V-ATPases caused by the second mutation. Thus RAVE is the first isoform-specific V-ATPase assembly factor.     

RAVE is essential for the efficient assembly of the C subunit with the vacuolar H(+)-ATPase

The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V(1) and V(0) sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V(1) peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V(1) complexes, suggesting that RAVE may play a role in docking of the V(1) peripheral stalk to the V(0) complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V(1) subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V(1) and V(0) sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V(1)/V(0) complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V(1) peripheral stalk subunit interactions with V(0) during V-ATPase assembly.     

Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly

SCF ubiquitin ligases are composed of Skp1, Cdc53, Hrt1 and one member of a large family of substrate receptors known as F-box proteins (FBPs). Here we report the identification, using sequential rounds of epitope tagging, affinity purification and mass spectrometry, of 16 Skp1 and Cdc53-associated proteins in budding yeast, including all components of SCF, 9 FBPs, Yjr033 (Rav1) and Ydr202 (Rav2). Rav1, Rav2 and Skp1 form a complex that we have named 'regulator of the (H+)-ATPase of the vacuolar and endosomal membranes' (RAVE), which associates with the V1 domain of the vacuolar membrane (H+)-ATPase (V-ATPase). V-ATPases are conserved throughout eukaryotes, and have been implicated in tumour metastasis and multidrug resistance, and here we show that RAVE promotes glucose-triggered assembly of the V-ATPase holoenzyme. Previous systematic genome-wide two-hybrid screens yielded 17 proteins that interact with Skp1 and Cdc53, only 3 of which overlap with those reported here. Thus, our results provide a distinct view of the interactions that link proteins into a comprehensive cellular network.     

The Ran GTPase cycle is required for yeast nuclear pore complex assembly

Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase-activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.     

Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum

The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V₁ sector catalyzes ATP hydrolysis and the V₀ sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V₀ assembly. We have discovered a fifth V₀ assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V₀ assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V₀ assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V₀–Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V₀ subunits c, c′, and c″. On assembly of the remaining three V₀ subunits (a, d, and e) into the V₀ complex, Voa1p dissociates from the now fully assembled V₀–Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V₀ assembly in the ER, but then it dissociates before exit of the V₀–Vma21p complex from the ER for transport to the Golgi compartment.     

The extreme C terminus of presenilin 1 is essential for gamma-secretase complex assembly and activity

The gamma-secretase complex catalyzes the cleavage of the amyloid precursor protein in its transmembrane domain resulting in the formation of the amyloid beta-peptide and the cytoplasmic APP intracellular domain. The active gamma-secretase complex is composed of at least four subunits: presenilin (PS), nicastrin, Aph-1, and Pen-2, where the presence of all components is critically required for gamma-cleavage to occur. The PS proteins are themselves subjected to endoproteolytic cleavage resulting in the generation of an N-terminal and a C-terminal fragment that remain stably associated as a heterodimer. Here we investigated the effects of modifications on the C terminus of PS1 on PS1 endoproteolysis, gamma-secretase complex assembly, and activity in cells devoid of endogenous PS. We report that certain mutations and, in particular, deletions of the PS1 C terminus decrease gamma-secretase activity, PS1 endoproteolysis, and gamma-secretase complex formation. We demonstrate that the N- and C-terminal PS1 fragments can associate with each other in mutants having C-terminal truncations that cause loss of interaction with nicastrin and Aph-1. In addition, we show that the C-terminal fragment of PS1 alone can mediate interaction with nicastrin and Aph-1 in PS null cells expressing only the C-terminal fragment of PS1. Taken together, these data suggest that the PS1 N- and C-terminal fragment intermolecular interactions are independent of an association with nicastrin and Aph-1, and that nicastrin and Aph-1 interact with the C-terminal part of PS1 in the absence of an association with full-length PS1 or the N-terminal fragment.     

The gene order in the nuo-operon is not essential for the assembly of E. coli complex I

Energy-converting NADH: ubiquinone oxidoreductase, respiratory complex I, plays an important role in cellular energy metabolism. Bacterial complex I is generally composed of 14 different subunits, seven of which are membranous and the other seven are globular proteins. They are encoded by the nuo-operon, whose gene order is strictly conserved in bacteria. The operon starts with nuoA encoding a membranous subunit followed by genes encoding globular subunits. To test the idea that NuoA acts as a seed to initiate the assembly of the complex in the membrane, we generated mutants that either lacked nuoA or contain nuoA at a different position within the operon. To enable the detection of putative assembly intermediates, the globular subunit NuoF and the membranous subunit NuoM were individually decorated with the fluorescent protein mCherry. Deletion of nuoA led to the assembly of an inactive complex in the membrane containing NuoF and NuoM. Re-arrangement of nuoA within the nuo-operon led to a slightly diminished amount of complex I in the membrane that was fully active. Thus, nuoA but not its distinct position in the operon is required for the assembly of E. coli complex I. Furthermore, we detected a previously unknown assembly intermediate in the membrane containing NuoM that is present in greater amounts than complex I.     

The essential Smp3 protein is required for addition of the side-branching fourth mannose during assembly of yeast glycosylphosphatidylinositols

The major glycosylphosphatidylinositols (GPIs) transferred to protein in mammals and trypanosomes contain three mannoses. In Saccharomyces cerevisiae, however, the GPI transferred to protein bears a fourth, alpha1,2-linked Man on the alpha1,2-Man that receives the phosphoethanolamine (EthN-P) moiety through which GPIs become linked to protein. We report that temperature-sensitive smp3 mutants accumulate a GPI containing three mannoses and that smp3 is epistatic to the gpi11, gpi13, and gaa1 mutations, which normally result in the accumulation of Man(4)-GPIs, including the presumed substrate for the yeast GPI transamidase. The Smp3 protein, which is encoded by an essential gene, is therefore required for addition of the fourth Man to yeast GPI precursors. The finding that smp3 prevents the formation of the Man(4)-GPI that accumulates when addition of EthN-P to Man-3 is blocked in a gpi13 mutant suggests that the presence of the fourth Man is important for transfer of EthN-P to Man-3 of yeast GPIs. The Man(3)-GPI that accumulates in smp3 is a mixture of two dominant isoforms, one bearing a single EthN-P side branch on Man-1, the other with EthN-P on Man-2, and these isoforms can be placed in separate arms of a branched GPI assembly pathway. Smp3-related proteins are encoded in the genomes of Schizosaccharomyces pombe, Candida albicans, Drosophila melanogaster, and Homo sapiens and form a subgroup of a family of proteins, the other groups of which are defined by the Pig-B(Gpi10) protein, which adds the third GPI mannose, and by the Alg9 and Alg12 proteins, which act in the dolichol pathway for N-glycosylation. Because Man(4)-containing GPI precursors are normally formed in yeast and Plasmodium falciparum, whereas addition of a fourth Man during assembly of mammalian GPIs is rare and not required for GPI transfer to protein, Smp3p-dependent addition of a fourth Man represents a target for antifungal and antimalarial drugs.     

Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p

How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.     

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.     

Pyruvate carboxylase is an essential protein in the assembly of yeast peroxisomal oligomeric alcohol oxidase

Hansenula polymorpha ass3 mutants are characterized by the accumulation of inactive alcohol oxidase (AO) monomers in the cytosol, whereas other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. These mutants also have a glutamate or aspartate requirement on minimal media. Cloning of the corresponding gene resulted in the isolation of the H. polymorpha PYC gene that encodes pyruvate carboxylase (HpPyc1p). HpPyc1p is a cytosolic, anapleurotic enzyme that replenishes the tricarboxylic acid cycle with oxaloacetate. The absence of this enzyme can be compensated by addition of aspartate or glutamate to the growth media. We show that HpPyc1p protein but not the enzyme activity is essential for import and assembly of AO. Similar results were obtained in the related yeast Pichia pastoris. In vitro studies revealed that HpPyc1p has affinity for FAD and is capable to physically interact with AO protein. These data suggest that in methylotrophic yeast pyruvate carboxylase plays a dual role in that, besides its well-characterized metabolic function as anapleurotic enzyme, the protein fulfils a specific role in the AO sorting and assembly process, possibly by mediating FAD-binding to AO monomers.     

Cytochrome c oxidase subunit IV is essential for assembly and respiratory function of the enzyme complex

Cytochrome c oxidase or complex IV, catalyzes the final step in mitochondrial electron transfer chain, and is regarded as one of the major regulation sites for oxidative phosphorylation. This enzyme is controlled by both nuclear and mitochondrial genomes. Among its 13 subunits, three are encoded by mitochondrial DNA and ten by nuclear DNA. In this work, an RNA interference approach was taken which led to the generation of mouse A9 cell derivatives with suppressed expression of nuclear-encoded subunit IV (COX IV) of this complex. The amounts of this subunit are decrease by 86% to 94% of normal level. A detail biosynthetic and functional analysis of several cell lines with suppressed COX IV expression revealed a loss of assembly of cytochrome c oxidase complex and, correspondingly, a reduction in cytochrome c oxidase-dependent respiration and total respiration. Furthermore, dysfunctional cytochrome c oxidase in the cells leads to a compromised mitochondrial membrane potential, a decreased ATP level, and failure to grow in galactose medium. Interestingly, suppression of COX IV expression also sensitizes the cells to apoptosis. These observations provide the evidence of the essential role of the COX IV subunit for a functional cytochrome c oxidase complex and also demonstrate a tight control of cytochrome c oxidase over oxidative phosphorylation. Finally, our results further shed some insights into the pathogenic mechanism of the diseases caused by dysfunctional cytochrome c oxidase complex.     

beta-subunit assembly is essential for the correct packing and the stable membrane insertion of the H,K-ATPase alpha-subunit

The alpha-subunits of H,K-ATPase (HKAalpha) and Na,K-ATPase require a beta-subunit for maturation. We investigated the role of the beta-subunit in the membrane insertion and stability of the HKAalpha expressed in Xenopus oocytes. Individual membrane segments M1, M2, M3, M4, and M9 linked to a glycosylation reporter act as signal anchor (SA) motifs, and M10 acts as a partial stop transfer motif. In combined HKAalpha constructs, M2 acts as an efficient stop transfer sequence, and M3 acts as a SA sequence. However, M5 and M9 have only partial SA function, and M7 has no SA function. Consistent with the membrane insertion properties of segments in combined alpha constructs, M1-3 alpha-proteins are resistant to cellular degradation, and M1-5 up to M1-10 alpha-proteins are not resistant to cellular degradation. However, co-expression with beta-subunits increases the membrane insertion of M9 in a M1-9 alpha-protein and completely protects M1-10 alpha-proteins against cellular degradation. Our results indicate that HKAalpha N-terminal (M1-M4) membrane insertion and stabilization are mediated by intrinsic molecular characteristics; however, the C-terminal (M5-M10) membrane insertion and thus the stabilization of the entire alpha-subunit depend on intramolecular and intermolecular beta-subunit interactions that are similar but not identical to data obtained for the Na,K-ATPase alpha-subunit.     

YfiO stabilizes the YaeT complex and is essential for outer membrane protein assembly in Escherichia coli

Recent advances in the study of bacterial membranes have led to the identification of a multicomponent YaeT complex in the outer membrane (OM) of Gram-negative bacteria that is involved in the targeting and folding of beta-barrel outer membrane proteins (OMPs). In Escherichia coli, this complex consists of an essential OMP, YaeT, and three OM lipoproteins, YfgL, NlpB and YfiO. YfiO is the only essential lipoprotein component of the complex. We show that this lipoprotein is required for the proper assembly and/or targeting of OMPs to the OM but not the assembly of lipopolysaccharides (LPS). Depletion of YfiO causes similar phenotypes as does the depletion of YaeT, and we conclude that YfiO plays a critical role in YaeT-mediated OMP folding. We demonstrate that YfiO and YfgL directly interact with YaeT in vitro, while NlpB interacts directly with YfiO. Genetic analysis verifies the importance of YfiO and its interactions with NlpB in maintaining the functional integrity of the YaeT complex.     

The polyubiquitin gene is essential for meiosis in fission yeast

We isolated a novel sporulation-deficient mutant of Schizosaccharomyces pombe. The mutant did not have a mitotic growth defect but aborted meiosis at the first or the second division with condensed chromosomes that failed to separate, abnormal spindle(s), and disintegrated spindle pole bodies (SPBs). During the first division, the centromeres were pulled to near the spindle poles but condensed divalent chromosomes remained at the center. The failure to proceed to anaphase was also observed during a time-lapse recording of a SPB protein tagged with green fluorescent protein. The polyubiquitin gene ubi4(+), which encoded eight ubiquitins fused in tandem, complemented this mutant. The mutation, an A to G substitution, was identified within the ubi4(+) gene at the ATG initiation codon. Disruption of the ubi4(+) gene produced the same phenotypes. The ubi4(+) mRNA was strongly induced for meiosis. However, ubiquitin increases only slightly, suggesting that the role of the polyubiquitin gene is to supply ubiquitin that is consumed by unidentified mechanisms. Before the ubi4 mutant cells entered meiosis, ubiquitin was greatly decreased indicating that shortage of ubiquitin caused abortion of meiosis. This work provides insights for the role of polyubiquitin gene and importance of ubiquitination in SPB integrity at the meiotic divisions.     

The fission yeast pleckstrin homology domain protein Spo7 is essential for initiation of forespore membrane assembly and spore morphogenesis

Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM.     

The SUMO protease SENP6 is essential for inner kinetochore assembly

We have analyzed the mitotic function of SENP6, a small ubiquitin-like modifier (SUMO) protease that disassembles conjugated SUMO-2/3 chains. Cells lacking SENP6 showed defects in spindle assembly and metaphase chromosome congression. Analysis of kinetochore composition in these cells revealed that a subset of proteins became undetectable on inner kinetochores after SENP6 depletion, particularly the CENP-H/I/K complex, whereas other changes in kinetochore composition mimicked defects previously reported to result from CENP-H/I/K depletion. We further found that CENP-I is degraded through the action of RNF4, a ubiquitin ligase which targets polysumoylated proteins for proteasomal degradation, and that SENP6 stabilizes CENP-I by antagonizing RNF4. Together, these findings reveal a novel mechanism whereby the finely balanced activities of SENP6 and RNF4 control vertebrate kinetochore assembly through SUMO-targeted destabilization of inner plate components.