Yeast vacuoles and membrane fusion pathways

Selective membrane fusion underlies subcellular compartmentation, cell growth, neurotransmission and hormone secretion. Its fundamental mechanisms are conserved among organelles, tissues and organisms. As befits a conserved process, reductionism led to its study in microorganisms. Homotypic fusion of the vacuole of Saccharomyces cerevisiae is particularly accessible to study as vacuoles are readily visualized, there is a rapid and quantitative in vitro assay of vacuole fusion, and the genetics and genomics of this organism and of vacuole fusion are highly advanced. Recent progress is reviewed in the context of general questions in the membrane fusion field.     

The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles

The V-ATPase V(0) sector associates with the peripheral V(1) sector to form a proton pump. V(0) alone has an additional function, facilitating membrane fusion in the endocytic and late exocytic pathways. V(0) contains a hexameric proteolipid cylinder, which might support fusion as proposed in proteinaceous pore models. To test this, we randomly mutagenized proteolipids. We recovered alleles that preserve proton translocation, normal SNARE activation and trans-SNARE pairing but that impair lipid and content mixing. Critical residues were found in all subunits of the proteolipid ring. They concentrate within the bilayer, close to the ring subunit interfaces. The fusion-impairing proteolipid substitutions stabilize the interaction of V(0) with V(1). Deletion of the vacuolar v-SNARE Nyv1 has the same effect, suggesting that both types of mutations similarly alter the conformation of V(0). Also covalent linkage of subunits in the proteolipid cylinder blocks vacuole fusion. We propose that a SNARE-dependent conformational change in V(0) proteolipids might stimulate fusion by creating a hydrophobic crevice that promotes lipid reorientation and formation of a lipidic fusion pore.     

The ubiquitin-proteasome system

The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first ‘tagged’ by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.     

The ubiquitin-proteasome pathway regulates claudin 5 degradation

The tight junctions (TJs) form continuous intracellular contacts, which help create selective barriers in epithelial and endothelial cell layers. The structures created by the TJs are very dynamic and can be rapidly remodeled in response to physiological and pathological signals. Claudin 5 is a membranal TJ protein which plays a critical role in determining the permeability of endothelial barriers. We describe the regulation of claudin 5 degradation by the ubiquitin-proteasome system (UPS). Our results indicate that claudin 5 has a relatively short half-life and can be polyubiquitinated on lysine 199. This ubiquitination appears to trigger the proteasome-dependent degradation of claudin 5. Other mechanisms also seem to be involved in the post-translational regulation of claudin 5, including a ubiquitin-independent and probably indirect lysosomal-dependent pathway. These findings provide evidence for the involvement of the UPS in the regulation of claudin 5 levels, and set the stage for further research to determine the involvement of this pathway in the modulation of the properties of TJs and cell-layer barriers.     

The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion

The farnesylated SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport.     

Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P(2)) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P(2) as approximately 6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P(2) soluble NSF attachment protein receptor (SNARE)-dependent liposome fusion is inhibited. Inhibition by PI 4,5-P(2) likely results from its intrinsic positive curvature-promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P(2), suggesting that syntaxin sequesters PI 4,5-P(2) to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P(2), we test a PI 4,5-P(2)-binding priming factor required for vesicle exocytosis. Ca(2+)-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P(2) dependent. These results indicate that PI 4,5-P(2) regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.     

Bem1p is a positive regulator of the homotypic fusion of yeast vacuoles

Docked vacuoles are believed to undergo rapid lipid mixing during hemifusion and then a slow, rate-limiting completion of fusion and mixing of lumenal contents. Previous genomic analysis has suggested that Bem1p, a scaffold protein critical for cell polarity, may support vacuole fusion. We now report that bem1Delta strains have fragmented vacuoles (vps class B and C). During in vitro fusion reactions, vacuoles from bem1Delta strains showed a strong reduction in the rate of lipid mixing when compared with vacuoles from the BEM1 parent. The reduction in the overall rate of fusion with bem1Delta vacuoles was modest, consistent with lipid mixing as a non-rate-limiting step in the pathway. Although the fusion of either BEM1 (wild-type) or bem1Delta vacuoles is stimulated by recombinant Bem1p, the lipid mixing of docked bem1Delta vacuoles is highly dependent on rBem1p under certain reaction conditions. Bem1p-stimulated lipid mixing is blocked by well characterized fusion inhibitors including lipid ligands and antibodies to Ypt7p, Vps33p, and Vam3p. Although full-length Bem1p is required for maximal stimulation, a truncation mutant comprising the SH3 domains and the Phox homology (PX) domain retains modest stimulatory activity. In contrast to an earlier report (Han, B. K., Bogomolnaya, L. M., Totten, J. M., Blank, H. M., Dangott, L. J., and Polymenis, M. (2005) Genes Dev. 19, 2606-2618), we did not find phosphorylation of Bem1p at Ser-72 to be required for Bem1p-stimulated fusion. Taken together, Bem1p is a positive regulator of lipid mixing during vacuole hemifusion and fusion.     

Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement

Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Delta growth defect selective for vacuole function. VRP1 encodes verprolin, an actin-binding protein that colocalizes to vacuoles. The vrp1Delta mutant has fragmented vacuoles in vivo and isolated vacuoles do not fuse in vitro, indicative of a Vrp1p requirement for membrane fusion. ERG6 overexpression rescues vrp1Delta vacuole fusion in a cytosol-dependent manner. Cytosol prepared from the vrp1Delta strain remains active; therefore, cytosol is not resupplying Vrp1p. Las17p (Vrp1p functional partner) antibodies, which inhibit wild-type vacuole fusion, do not inhibit the fusion of vacuoles from the vrp1Delta-ERG6 overexpression strain. Vacuole-associated actin turnover is decreased in the vrp1Delta strain, but recovered by ERG6 overexpression linking sterol enrichment to actin remodeling. Therefore, the Vrp1p/Las17p requirement for membrane fusion is bypassed by increased sterols, which promotes actin remodeling as part the membrane fusion mechanism.     

tRNA import into yeast mitochondria is regulated by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, one of two cytosolic lysine-tRNAs is partially imported into mitochondria. We demonstrate that three components of the ubiquitin/26S proteasome system (UPS), Rpn13p, Rpn8p and Doa1p interact with the imported tRNA and with the essential factor of its mitochondrial targeting, pre-Msk1p. Genetic and biochemical assays demonstrate that UPS plays a dual regulatory role, since the overall inhibition of cellular proteasome activity reduces tRNA import, while specific depletion of Rpn13p or Doa1p increases it. This result suggests a functional link between UPS and tRNA mitochondrial import in yeast and indicates on the existence of negative and positive import regulators.     

The nuclear ubiquitin-proteasome system degrades MyoD

Many short-lived nuclear proteins are targeted for degradation by the ubiquitin-proteasome pathway. The role of the nucleus in regulating the turnover of these proteins is not well defined, although many components of the ubiquitin-proteasome system are localized in the nucleus. We have used nucleoplasm from highly purified HeLa nuclei to examine the degradation of a physiological substrate of the ubiquitin-proteasome system (MyoD). In vitro studies using inhibitors of the system demonstrate MyoD is degraded via the ubiquitin-proteasome pathway in HeLa nucleoplasm. Purified nucleoplasm in vitro also supports the generation of high molecular mass MyoD-ubiquitin adducts. In addition, in vivo studies, using leptomycin B to inhibit nuclear export, demonstrate that MyoD is degraded in HeLa cells by the nuclear ubiquitin-proteasome system.     

The ubiquitin-proteasome system in Alzheimer's disease

Accumulation of proteins is a recurring event in many neurodegenerative diseases, including Alzheimer's disease (AD). Evidence has suggested that protein accumulation may result from a dysfunction in the ubiquitin proteasome system (UPS). Indeed, there is clear genetic and biochemical evidence of an involvement of the ubiquitin proteasome system in AD. This review summarizes the data supporting an involvement of the UPS in the pathogenesis of AD, focusing on the data showing the relationship between Abeta and tau, the two hallmark lesions of AD, and the UPS.     

The ubiquitin-proteasome system in colorectal cancer

The proteasome is a multiprotein complex that regulates the stability of hundreds of cellular proteins and thus, it is implicated in virtually all cellular functions. Most of the time, to be recognized and processed by the proteasome, a protein has to be linked to a chain of ubiquitin molecules. Cell proliferation, apoptosis, angiogenesis and motility, processes with particular importance for carcinogenesis are regulated by the ubiquitin-proteasome system (UPS). In colorectal epithelium, UPS plays a role in the regulation of the Wnt/beta-catenin/APC/TCF4 signaling which regulates proliferation of colorectal epithelial cells in the bottom of the crypts and the inhibition of this proliferation as cells move towards colon villi tips. In most colorectal cancers APC (Adenomatous Polyposis Coli) disabling mutations interfere with the ability of the proteasome to degrade beta-catenin leading to uninhibited cell proliferation. Other key molecules in colorectal carcinogenesis such as p53, Smad4 and components of the k-ras pathways are also regulated by the UPS. In this review I discuss the role of UPS in colorectal carcinogenesis and colorectal cancer prognosis and aspects of its inhibition for therapeutic purposes.     

The ubiquitin-proteasome system in cardiac dysfunction

Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin-proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease.     

The plasma membrane proteins Prm1 and Fig1 ascertain fidelity of membrane fusion during yeast mating

As for most cell-cell fusion events, the molecular details of membrane fusion during yeast mating are poorly understood. The multipass membrane protein Prm1 is the only known component that acts at the step of bilayer fusion. In its absence, mutant mating pairs lyse or arrest in the mating reaction with tightly apposed plasma membranes. We show that deletion of FIG 1, which controls pheromone-induced Ca(2+) influx, yields similar cell fusion defects. Although extracellular Ca(2+) is not required for efficient cell fusion of wild-type cells, cell fusion in prm1 mutant mating pairs is dramatically reduced when Ca(2+) is removed. This enhanced fusion defect is due to lysis. Time-lapse microscopy reveals that fusion and lysis events initiate with identical kinetics, suggesting that both outcomes result from engagement of the fusion machinery. The yeast synaptotagmin orthologue and Ca(2+) binding protein Tcb3 has a role in reducing lysis of prm1 mutants, which opens the possibility that the observed role of Ca(2+) is to engage a wound repair mechanism. Thus, our results suggest that Prm1 and Fig1 have a role in enhancing membrane fusion and maintaining its fidelity. Their absence results in frequent mating pair lysis, which is counteracted by Ca(2+)-dependent membrane repair.     

Characterization of a specific transport system for arginine in isolated yeast vacuoles.

The transport of L-arginine was studied in isolated vacuoles of Saccharomyces cerevisiae. A centrifugation method allowed rapid separation of the fragile vacuoles from the incubation media so that initial uptake rates of [14C]arginine could be measured. Labelled arginine added to the medium was accumulated in the isolated vacuoles; it was found to exchange specifically with the arginine already present in the vacuoles. Such an exchange did not take place in intact spheroplasts. The pH dependence of the arginine transport in the vacuoles was tested. As the vacuoles are unstable in the pH range of optimal transport activity (pH above 7.0), the pH optimum of the transport reaction could not be determined. From the temperature dependence, the apparent energy of activation was calculated to be 9800 cal/mol. Arginine transport shows saturation kinetics with an apparent Km of 30 muM in the isolated vacuoles, and of 1.5 muM in the spheroplasts. Competition experiments with amino acids and arginine analogues demonstrated that the arginine transport in both vacuoles and spheroplasts, is highly specific. The two systems, however, were shown to have distinct specificities. The inhibition of vacuolar L-arginine transport by D-arginine, L-histidine, and L-canavanine was competitive with apparent Ki values of 60 muM, 400 muM and 600 muM respectively.     

Cdc42 regulates multiple membrane traffic events in fission yeast

Fission yeast Cdc42 regulates polarized growth and is involved in For3 formin activation and actin cable assembly. We show here that a thermosensitive strain carrying the cdc42L160S allele has membrane traffic defects independent of the actin cable defects. This strain has decreased acid phosphatase (AP) secretion, intracellular accumulation of vesicles and fragmentation of vacuoles. In addition, the exocyst is not localized to the tips of these cells. Overproduction of the scaffold protein Pob1 suppressed cdc42L160S thermosensitive growth and restored exocyst localization and AP secretion. The GTPase Rho3 also suppressed cdc42L160S thermosensitivity, restored exocyst localization and AP secretion. However, Rho3 did not restore the actin cables in these cells as Pob1 does. Similarly, overexpression of psy1(+) , coding a syntaxin (t-SNARE) homolog, or of ypt2(+) , coding an SEC4 homolog in fission yeast, rescued growth at high temperature but did not restore actin cables, nor the exocyst-polarized localization. cdc42L160S cells also have defects in vacuole formation that were rescued by Pob1, Rho3 and Psy1. All together, we propose that Cdc42 and the scaffold Pob1 are required for membrane trafficking and fusion, contributing to polarized secretion, endosome recycling, vacuole formation and growth.     

Phosphorelay-regulated degradation of the yeast Ssk1p response regulator by the ubiquitin-proteasome system

In Saccharomyces cerevisiae, a phosphorelay signal transduction pathway composed of Sln1p, Ypd1p, and Ssk1p, which are homologous to bacterial two-component signal transducers, is involved in the osmosensing mechanism. In response to high osmolarity, the phosphorelay system is inactivated and Ssk1p remains unphosphorylated. Unphosphorylated Ssk1p binds to and activates the Ssk2p mitogen-activated protein (MAP) kinase kinase kinase, which in turn activates the downstream components of the high-osmolarity glycerol response (HOG) MAP kinase cascade. Here, we report a novel inactivation mechanism for Ssk1p involving degradation by the ubiquitin-proteasome system. Degradation is regulated by the phosphotransfer from Ypd1p to Ssk1p, insofar as unphosphorylated Ssk1p is degraded more rapidly than phosphorylated Ssk1p. Ubc7p/Qri8p, an endoplasmic reticulum-associated ubiquitin-conjugating enzyme, is involved in the phosphorelay-regulated degradation of Ssk1p. In ubc7Delta cells in which the degradation is hampered, the dephosphorylation and/or inactivation process of the Hog1p MAP kinase is delayed compared with wild-type cells after the hyperosmotic treatment. Our results indicate that unphosphorylated Ssk1p is selectively degraded by the Ubc7p-dependent ubiquitin-proteasome system and that this mechanism downregulates the HOG pathway after the completion of the osmotic adaptation.