The ego-1 gene is the first example of a gene encoding an RdRP-related protein with an essential developmental function. The ego-1 gene is also required for a robust response to RNA interference by certain genes. Hence, a protein required for germ-line development in C. elegans may be a component of the RNA interference/PTGS machinery.

Vacuolar proton-translocating ATPases (V-ATPases) 1 are highly conserved proton pumps responsible for acidification of organelles such as the lysosome/vacuole, Golgi apparatus, and endosomes in all eukaryotic cells (1-3). In some cells, VATPases are also present at high levels at the plasma membrane, where they pump protons from the cytosol out of the cell (2, 4). In all of these locations and in organisms ranging from yeast to humans, V-ATPases have a very similar structure and subunit composition. They are comprised of 13 or 14 subunits arranged as a complex of cytosolic peripheral membrane subunits containing the sites of ATP hydrolysis, the V 1 sector, attached to a membrane complex containing the proton pore, the V o sector. ATP-driven proton transport occurs only when the two sectors are structurally and functionally coupled. Free V 1 sectors do not catalyze hydrolysis of MgATP, the physiological substrate, and free V o sectors do not appear to form open proton pores (5-7).The assembly pathways for V-ATPases are complicated and incompletely understood. Both mammalian cells and yeast contain free V 1 and V o sectors in vivo (8 -10), and yeast mutants lacking one subunit of either sector are able to assemble the other sector (10). Yet there is evidence that the major pathway for biosynthetic assembly of V-ATPases does not involve independent assembly of free V 1 and V o sectors followed by attachment of the two sectors. Instead, pulse-chase studies indicate very early association of V 1 and V o sector subunits followed by addition of subunits from both sectors (11). Definition of assembly pathways was further complicated by the observation that fully assembled V-ATPases could rapidly and reversibly disassemble into free V 1 and V o sectors (12, 13). Disassembly of V-ATPases in yeast and insects, the two systems in which the process is best characterized, occurs in response to low extracellular glucose concentrations. Reversible disassembly is believed to be an important regulatory mechanism; disassembly of V-ATPases conserves ATP under conditions of nutrient limitation by silencing the ATPase activity of the enzyme and reassembly rapidly reactivates the pump with no need for new protein synthesis (14,15).The signaling pathways connecting V-ATPase assembly with extracellular glucose concentration have proven to be somewhat elusive. Many of the pathways that signal glucose availability in yeast cells do not appear to be involved in modulating the assembly state of the V-ATPase (16). Recently, a new player in V-ATPase assembly was identified. Seol et al. (17) identified a Skp1p-containing complex they called RAVE, regulator of the ATPase of vacuolar and endosomal membranes, through affinity chromatography to detect Skp1p binding partners, and subsequently showed that RAVE also bound at least four of the V 1 subunits of the yeast V-ATPase. The RAVE complex contains three members, Rav1p, Rav2p, and Skp1p. RAV1 and RAV2 were previously uncharacterized yeast open reading frames, but RAV1 has homologues in all euk...

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, ‫0074ف‬ yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl 2 . In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36⌬ mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34⌬, vps15⌬, vps45⌬, and vps16⌬, caused both VmaϪ growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1⌬ mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a VmaϪ growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the VmaϪ growth phenotype.

In yeast cells, subunit a of the vacuolar proton pump (VATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1⌬ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1⌬ and stv1⌬ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.Vacuolar proton-translocating ATPases (V-ATPases) 3 acidify multiple organelles, including mammalian lysosomes, plant and fungal vacuoles, the Golgi apparatus, endosomes, and regulated secretory granules. Through their effects on organelle acidification, V-ATPases impact numerous cellular processes including protein sorting, macromolecular degradation, cytosolic pH and ion homeostasis, and nutrient storage and mobilization (1, 2). Consistent with these diverse roles, complete loss of V-ATPase function is lethal in most organisms. Fungi, however, can tolerate a complete loss of V-ATPase function, and Saccharomyces cerevisiae has emerged as a major model system for mechanistic studies of V-ATPases (3). Yeast mutants lacking V-ATPase activity (vma mutants) show a well defined set of Vma Ϫ growth phenotypes, including sensitivity to high extracellular pH, high Ca 2ϩ concentrations, and heavy metals (4).V-ATPases are highly conserved both at the level of individual subunit sequences and at an overall structural level. A complex of peripheral membrane subunits containing the sites of ATP hydrolysis, V 1 , is attached to an integral membrane complex, V o , containing the proton pore. In higher eukaryotes, many of the subunits are present as multiple isoforms, encoded as multiple genes and/or splice variants (5). These subunit isoforms exhibit tissue-specific expression and/or organelle-specific localization, and in some cases, impart different biochemical characteristics on V-ATPases, possibly tuning their activity to the requirements of different locales (2). Subunit a of the V o sector is present as multiple isoforms in many organisms. Humans have four different subunit a genes (...

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.Vacuolar proton-translocating ATPases (V-ATPases) 2 are conserved in all eukaryotic cells where they function to acidify internal organelles such as the lysosome/vacuole, Golgi apparatus, secretory vesicles, and endosomes. V-ATPases couple hydrolysis of cytoplasmic ATP to transport of protons from the cytosol into intracellular compartments. Organelle acidification is essential for a wide range of cellular processes including protein sorting in the biosynthetic and endocytic pathways; protein processing, activation, and degradation; cellular ion homeostasis; and coupled transport of small molecules (1-4). V-ATPases also have been identified in the plasma membrane of certain specialized cells where they pump protons from the cytosol out of the cell (1, 5). The structure and subunit composition of V-ATPases is very similar in all organisms from yeast to humans. They are multisubunit complexes composed of two domains. The V 1 domain is a peripheral cytoplasmic complex composed of eight different subunits (subunits A-H), and it contains the sites of ATP hydrolysis. The V 0 domain is an integral membrane complex that is composed of six different subunits (subunits a, d, e, c, cЈ, and cЉ). It comprises the proton pore. The V 1 and V 0 domains must be structurally and functionally coupled for ATP-driven proton translocation to occur. V 1 complexes that are not attached to V 0 at the membrane cannot hydrolyze MgATP, and V 0 complexes in the membrane that are not attached to V 1 are not able to transport protons (6, 7).The biosynthetic assembly pathway for V-ATPases is not completely understood. Free V 1 and V 0 complexes exist in vivo in both yeast and mammalian cells (8 -11). Independent ...