The Vacuole Import and Degradation Pathway Utilizes Early Steps of Endocytosis and Actin Polymerization to Deliver Cargo Proteins to the Vacuole for Degradation

When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.     

Transport of a Large Oligomeric Protein by the Cytoplasm to Vacuole Protein Targeting Pathway

Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.     

The TOR Complex 1 Is Distributed in Endosomes and in Retrograde Vesicles That Form from the Vacuole Membrane and Plays an Important Role in the Vacuole Import and Degradation Pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.     

Vacuole Biogenesis in Saccharomyces cerevisiae: Protein Transport Pathways to the Yeast Vacuole

Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.     

Identification of a Lysosomal Pathway Regulating Degradation of the Bone Morphogenetic Protein Receptor Type II

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease.     

蛋白质的选择性降解机制——2004年诺贝尔化学奖部分工作介绍

如何识别和选择性降解蛋白质是细胞生命过程中的重要环节.泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式.对于需要清除的蛋白质,通过其赖氨酸残基侧链ε-氨基连接多聚泛素链（降解标签）,继而在蛋白酶体中被降解.这种选择性降解机制对于维持蛋白质在细胞内含量的动态平衡起到了关键性作用.     

A Cytoplasm to Vacuole Targeting Pathway in P. pastoris

The cytoplasm-to-vacuole targeting (Cvt) pathway of Saccharomyces cerevisiae delivers aminopeptidase I (Ape1) from the cytosol to the vacuole, bypassing the normal secretory route. The Cvt pathway, although well-studied, was known only in S. cerevisiae. We demonstrate its existence in the methylotrophic yeast, Pichia pastoris, where it also delivers P. pastoris Ape1 (PpApe1) to the vacuole. Most proteins known to be required for the Cvt pathway in S. cerevisiae were, to the extent we found orthologs, also required in P. pastoris. The P. pastoris Cvt pathway differs, however, from that in S. cerevisiae, in that new proteins, such as PpAtg28 and PpAtg26, are involved. The discovery of a Cvt pathway in P. pastoris makes it an excellent model system for the dissection of autophagy-related pathways in a single organism and for the discovery of new Cvt pathway components.     

Identification of Kinesin-Like Protein on Golgi Vesicles of Pollen

Kinesin-like protein was identified on Golgi vesicles of pollen. At the tip of pollen tube of Nicotiana alata, the vesicle-like particles were recognized by monoclonal antibody against the kinesin heavy chain from bovine brain (K71s23). The Glogi vesicles isolated from the pollens of Corylus avellana by discontinious sucrose gradient ultracentrifugation, could be recognized as antikinesin, based on immuno-gold labelling. Results from SDS-PAGE and western blot, showed that the 100 kD polypeptides on Golgi vesicles were the major polypeptides of kinesin-like protein.     

Protein Aggregation Profile of the Bacterial Cytosol

Background

Protein misfolding is usually deleterious for the cell, either as a consequence of the loss of protein function or the buildup of insoluble and toxic aggregates. The aggregation behavior of a given polypeptide is strongly influenced by the intrinsic properties encoded in its sequence. This has allowed the development of effective computational methods to predict protein aggregation propensity.

Methodology/Principal Findings

Here, we use the AGGRESCAN algorithm to approximate the aggregation profile of an experimental cytosolic Escherichia coli proteome. The analysis indicates that the aggregation propensity of bacterial proteins is associated with their length, conformation, location, function, and abundance. The data are consistent with the predictions of other algorithms on different theoretical proteomes.

Conclusions/Significance

Overall, the study suggests that the avoidance of protein aggregation in functional environments acts as a strong evolutionary constraint on polypeptide sequences in both prokaryotic and eukaryotic organisms.     

Evidence for Catalytic Roles for Plasmodium falciparum Aminopeptidase P in the Food Vacuole and Cytosol

The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 °C in the pH range 5.0–7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.Malaria remains one of the most deadly global infectious diseases with an estimated 500 million clinical cases and 2 million deaths annually (1, 2). Clinical manifestations of the disease arise as the protozoan malaria parasite replicates asexually within human erythrocytes. Five species of the genus Plasmodium infect humans. The cytoadherent properties of red blood cells infected with Plasmodium falciparum, coupled with the ability of the parasite to reach high parasitemia, make it the most virulent species. The emergence of strains of P. falciparum that are resistant to affordable anti-malarial drugs such as chloroquine has complicated efforts to manage malaria, and new drugs are urgently needed.Aminopeptidases catalyze the hydrolysis of amino acids from the amino termini of proteins and peptides. They participate in a wide range of biological processes, including peptide catabolism, protein maturation, antigen presentation on immune cells, and regulation of hormone activity. During the asexual erythrocytic replication cycle of the malaria parasite, aminopeptidases contribute to the catabolism of peptides generated by two major proteolytic pathways. One of these is initiated at the proteasome, a multifunctional protease that plays an important role in the turnover of ubiquitinated cellular proteins in the cytosol (3–5). In addition, the parasite transports host red blood cell cytosol (consisting primarily of hemoglobin) to an acidic degradative organelle, the food vacuole, where it is degraded in a proteasome-independent pathway (6, 7). As up to 75% of the host cell hemoglobin is catabolized during the intraerythrocytic cycle (8, 9), flux through the vacuolar pathway is substantial. Three aminopeptidases have been identified as key players in recycling amino acids from peptides generated by the proteasomal and vacuolar catabolic pathways: leucine aminopeptidase, aminopeptidase N (PfA-M1), and aminopeptidase P (10–14). The latter two enzymes have been found in the food vacuole and therefore may play a direct role in hemoglobin catabolism (11). An aspartyl aminopeptidase is also expressed in asexual stage parasites and hydrolyzes amino-terminal aspartyl and glutamyl substrates (15); however, disruption of its gene does not prevent efficient intraerythrocytic replication (11).Aminopeptidase P (APP)² homologs exhibit high specificity for proline in the second position of the substrate (the P1′ position in the nomenclature of Schechter and Berger (16)) and catalyze the hydrolysis of the X-Pro amide bond, where X is any aminoacyl residue (17). Because of the cyclic nature of the proline side chain, X-Pro-containing peptides are not easily accommodated in the active sites of broad specificity aminopeptidases (17). In mammals, three APP isozymes have been identified. APP1 is found in the cytosolic fraction of cell lysates and has been characterized from a variety of tissues (18–20). Although this enzyme has not, to our knowledge, been localized in intact cells, the apparent lack of specific targeting information is consistent with a role in cytosolic peptide turnover. Active cytosolic forms of APP have been reported in plants (21), fruit flies (22), the microsporidian parasite Encephalitozoon cuniculi (23), and in intestinal cells in Caenorhabditis elegans (24), where it is believed to play a role in the catabolism of peptides produced from ingested bacteria. Mammalian APP2 is a glycosylated ectoenzyme anchored into the membrane of endothelial and epithelial cells with a glycosylphosphatidylinositol attachment (25). The best characterized role of APP2 is the inactivation of the plasma hormone bradykinin, a nonapeptide, through cleavage of the Arg-Pro amino-terminal peptide bond (26, 27). Inhibition of APP2 potentiates the vasodilatory and cardioprotective properties of bradykinin, and APP2 has been considered a target for the development of cardiovascular drugs (28–30). A third isoform, APP3, has been identified in the human genome and may be a mitochondrial enzyme but has not yet been characterized (31). Prokaryotic APP homologs contribute to intracellular peptide turnover (32).P. falciparum aminopeptidase P (PfAPP) appears to be important for intraerythrocytic growth, as parasites with a disrupted PfAPP gene could not be isolated (11). We have previously localized a PfAPP-yellow fluorescent protein fusion to the food vacuole and the cytosol of the parasite (11). The cytosolic pool of PfAPP probably fulfills a role in peptide turnover and amino acid recycling that is orthologous to those of the cytosolic enzymes described above. In contrast, there is no report to our knowledge of an aminopeptidase P homolog functioning in an acidic environment akin to the malarial food vacuole. Moreover, characterization of mammalian aminopeptidase P homologs typically reveals a pH optimum of 7–8 with relatively little, if any, activity in the pH range 5.0–5.5 (18–20). Although we have previously detected PfAPP activity at acidic pH (11), the catalytic efficiency of the enzyme has not been characterized. Thus, at the outset of this study it was not clear whether PfAPP has a significant catalytic role in the food vacuole.Here we have localized untagged, native PfAPP in the parasite and have confirmed the dual cytosolic/vacuolar distribution of the enzyme. The domain organization, quaternary structure, and metal requirement of PfAPP were characterized. To evaluate the plausibility of a catalytic role for PfAPP at acidic and near-neutral pH, its stability in the pH range 5.0–7.5 was assessed and compared with that of human cytosolic APP1, an enzyme that does not, to our knowledge, have a physiological role in an acidic environment. The catalytic efficiency of PfAPP at a range of pH values was characterized with three X-Pro-containing peptides, two of which are found in the sequences of human α- and β-globin and therefore represent potentially physiological substrates.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Vac8p, a Vacuolar Protein with Armadillo Repeats, Functions in both Vacuole Inheritance and Protein Targeting from the Cytoplasm to Vacuole

During each cell cycle, the yeast vacuole actively partitions between mother and daughter cells. This process requires actin, profilin, an unconventional myosin (Myo2p), and Vac8p. A mutant yeast strain, vac8, is defective in vacuole inheritance, specifically, in early vacuole migration. Vac8p is a 64-kD protein found on the vacuole membrane, a site consistent with its role in vacuole inheritance. Both myristoylation and palmitoylation are required for complete Vac8p localization. Interestingly, whereas myristoylation of Vac8p is not required for vacuole inheritance, palmitoylation is essential. Thus, palmitoylation appears to play a more direct role in vacuole inheritance. Most of the VAC8 sequence encodes 11 armadillo (Arm) repeats. Arm repeats are thought to mediate protein–protein interactions, and many Arm proteins have multiple functions. This is also true for Vac8p. In addition to its role in early vacuole inheritance, Vac8p is required to target aminopeptidase I from the cytoplasm to the vacuole. Mutant analysis demonstrates that Vac8p functions separately in these two processes. Vac8p cosediments with actin filaments. Vac8p is related to β-catenin and plakoglobin, which connect a specific region of the plasma membrane to the actin cytoskeleton. In analogy, Vac8p may link the vacuole to actin during vacuole partitioning.     

Characterization of the Ubiquitylating Components of the Human Malaria Parasite's Protein Degradation Pathway

Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.     

Protein Transport from the Cytoplasm into the Vacuole

The fungal vacuole is integrally involved in various cellular processes that include protein and organellar degradation and recycling. The ability to sequester numerous hydrolases within the cell makes the hydrolytic capacity of the vacuole critical under certain environmental conditions. Accordingly, cellular constituents destined for degradation are delivered to the vacuole through the secretory pathway, by endocytosis and from the cytoplasm. Different mechanisms have evolved to accommodate these multiple transport pathways. Protein transport from the cytoplasm into the vacuole in particular relies on the dynamic nature of the vacuole membrane. This review describes recent research on this topic from yeast systems and points out the direction of future studies aimed at understanding this complex organelle. Received: 13 January/Revised: 9 March 1997     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.