Patricia M. Kane

Reversible Association between the V1 and V0 Domains of Yeast Vacuolar H+-ATPase Is an Unconventional Glucose-Induced Effect

ABSTRACT The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.

Regulation of V-ATPases by reversible disassembly

V‐ATPases consist of a complex of peripheral subunits containing catalytic sites for ATP hydrolysis, the V1 sector, attached to several membrane subunits containing a proton pore, the V0 sector. ATP‐driven proton transport requires structural and functional coupling of the two sectors, but in vivo, the interaction between the V1 and V0 sectors is dynamic and is regulated by extracellular conditions. Dynamic instability appears to be a general characteristic of V‐ATPases and, in yeast cells, the assembly state of V‐ATPases is governed by glucose availability. The structural and functional implications of reversible disassembly of V‐ATPases are discussed.

MUTATIONS IN THE CYS4 GENE PROVIDE EVIDENCE FOR REGULATION OF THE YEAST VACUOLAR H+-ATPASE BY OXIDATION AND REDUCTION IN VIVO

The vma41-1 mutant was identified in a genetic screen designed to identify novel genes required for vacuolar H+-ATPase activity in Saccharomyces cerevisiae. The VMA41 gene was cloned and shown to be allelic to theCYS4 gene. The CYS4 gene encodes the first enzyme in cysteine biosynthesis, and in addition to cysteine auxotrophy, cys4 mutants have much lower levels of intracellular glutathione than wild-type cells. cys4mutants display the pH-dependent growth phenotypes characteristic of vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidificationin vivo. The vacuolar proton-translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific activity is reduced (47% of wild type) and the activity is unstable. Addition of reduced glutathione to the growth medium complements the pH-dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity. TheCYS4 gene was deleted in a strain in which the catalytic site cysteine residue implicated in oxidative inhibition of the yeast V-ATPase has been mutagenized (Liu, Q., Leng, X.-H., Newman, P., Vasilyeva, E., Kane, P. M., and Forgac, M. (1997) J. Biol. Chem. 272, 11750–11756). This catalytic site point mutation suppresses the effects of the cys4 mutation. The data indicate that the acidification defect of cys4 mutants arises from inactivation of the vacuolar ATPase in the less reducing cytosol resulting from loss of Cys4p activity and provide the first evidence for the modulation of V-ATPase activity by the redox state of the environment in vivo.

Early Steps in Assembly of the Yeast Vacuolar H+-ATPase

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3–5 min, followed by addition of other Vosubunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vosubunits during or after exit from the endoplasmic reticulum.

Characterization of a Temperature-sensitive Yeast Vacuolar ATPase Mutant with Defects in Actin Distribution and Bud Morphology

The 27-kDa E subunit, encoded by theVMA4 gene, is a peripheral membrane subunit of the yeast vacuolar H+-ATPase. We have randomly mutagenized theVMA4 gene in order to examine the structure and function of the 27-kDa subunit. Cells lacking a functional VMA4 gene are unable to grow at pH > 7 or in elevated concentrations of CaCl2. Plasmid-borne, mutagenized vma4 genes were screened for failure to complement these phenotypes. Mutants producing Vma4 proteins detectable by immunoblot were selected; one (vma4–1 ts ) is temperature conditional, exhibiting the Vma− phenotype only at elevated temperature (37 °C). Sequencing revealed that a single point mutation, D145G, was responsible for the phenotypes of thevma4-1 ts allele. The unassembled 27-kDa subunit made in the vma4-1 ts cells is rapidly degraded, particularly at 37 °C, but can be protected from degradation by prior assembly into the V-ATPase complex. In purified vacuolar vesicles from the mutant cells, the peripheral subunits are localized to the vacuolar membrane at decreased levels and a comparably decreased level of ATPase activity (14% of the activity in wild-type vesicles) is observed. When vma4-1 ts mutant cells are shifted to pH 7.5 medium at 37 °C, the cells become enlarged and exhibit multiple large buds, elongated buds, and other abnormal morphologies, together with delocalization of actin and chitin, within 4 h. These phenotypes suggest connections between the vacuolar ATPase, bud morphology, and cytokinesis that had not been recognized previously.

Mutations in the Yeast KEX2 Gene Cause a Vma−-Like Phenotype: a Possible Role for the Kex2 Endoprotease in Vacuolar Acidification

ABSTRACT Mutants of Saccharomyces cerevisiae that lack vacuolar proton-translocating ATPase (V-ATPase) activity show a well-defined set of Vma− (stands for vacuolar membrane ATPase activity) phenotypes that include pH-conditional growth, increased calcium sensitivity, and the inability to grow on nonfermentable carbon sources. By screening based on these phenotypes and the inability ofvma mutants to accumulate the lysosomotropic dye quinacrine in their vacuoles, five new vma complementation groups (vma41 to vma45) were identified. TheVMA45 gene was cloned by complementation of the pH-conditional growth of the vma45-1 mutant strain and shown to be allelic to the previously characterized KEX2gene, which encodes a serine endoprotease localized to the late Golgi compartment. Both vma45-1 mutants and kex2 null mutants exhibit the full range of Vma− growth phenotypes and show no vacuolar accumulation of quinacrine, indicating loss of vacuolar acidification in vivo. However, immunoprecipitation of the V-ATPase from both strains under nondenaturing conditions revealed no defect in assembly of the enzyme, vacuolar vesicles isolated from akex2 null mutant showed levels of V-ATPase activity and proton pumping comparable to those of wild-type cells, and the V-ATPase complex purified from kex2 null mutants was structurally indistinguishable from that of wild-type cells. The results suggest that kex2 mutations exert an inhibitory effect on the V-ATPase in the intact cell but that the ATPase is present in the mutant strains in a fully assembled state, potentially capable of full enzymatic activity. This is the first time a mutation of this type has been identified.

Wild-type and Mutant Vacuolar Membranes Support pH-dependent Reassembly of the Yeast Vacuolar H+-ATPase in Vitro

Treatment of the yeast vacuolar proton-translocating ATPase (H+-ATPase) with 300 mM KI in the presence of 5 mM MgATP results in a 90% inhibition of ATPase activity accompanied by removal of at least five of the peripheral subunits of the enzyme from the membrane. Functional reassembly of the enzyme, as indicated by reattachment of the peripheral subunits and a partial (30-70%) recovery of ATPase activity, could be achieved by dialysis of the stripped wild-type membranes to remove the KI and MgATP, but proved to be strongly pH-dependent, with optimal reassembly and recovery of activity occurring after dialysis at pH 5.5. Vacuolar membranes isolated from vma2Δ mutants, which lack one of the peripheral subunits of the enzyme, do not contain any of the peripheral subunits but are shown to contain assembled membrane (Vo) complexes. The vma2Δ mutant vacuoles are demonstrated to be competent for attachment of KI-stripped peripheral subunits and reactivation of ATPase activity. The results indicate that previously assembled Vo complexes are capable of inducing assembly of the peripheral subunits, both with each other and with the membrane subunits, and of activating the ATPase activity that resides in the peripheral subunits in a pH-dependent manner.

Biosynthesis and regulation of the yeast vacuolar H+-ATPase.

The yeast V-ATPase is highly similar to V-ATPases of higher organismsand has proved to be a biochemically and genetically accessible model formany aspects of V-ATPase function. Like other V-ATPases, the yeast enzymeconsists of a complex of peripheral membrane proteins, the V1sector, attached to a complex of integral membrane subunits, theV0 sector. Multiple pathways for biosynthetic assembly of theenzyme appear to be available to cells containing a full complement ofsubunits and enzyme activity may be further controlled during biosynthesis bya protease activity localized to the late Golgi apparatus. Surprisingly, theassembled V-ATPase is not a static structure. Instead, fully assembledV1V0 complexes appear to exist in a dynamic equilibriumwith inactive cytosolic V1 and membrane-bound V0complexes and this equilibrium can be rapidly shifted in response to changesin carbon source. The reversible disassembly of the yeast V-ATPase may be anovel regulatory mechanism, common to V-ATPases, that works in vivoin coordination with many other regulatory mechanisms.

Energy powerhouses of cells come into focus

High-resolution structures reveal core design features of rotary ATP synthases and ATPases In every kingdom of life, rotary adenosine triphosphate (ATP) synthases and adenosine triphosphatases (ATPases) play key roles in cellular energy generation and release processes. In mitochondria, chloroplasts, and bacteria, F-type (F1Fo) ATP synthases synthesize ATP using energy from a proton gradient. They are also able to perform the reverse process, generating proton gradients by ATP hydrolysis. The related V-type (V1Vo) ATPases have similar structures and serve as proton pumps. Two articles in this issue report structures of membrane-embedded ATP synthases from yeast mitochondria [Srivastava et al., page 619, (1)] and spinach chloroplasts [Hahn et al., page 620, (2)]. Together with other recent structures, these articles define core design principles of rotary ATP synthases and ATPases but also highlight organism-specific differences.