Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast.

Becherer, Kathleen; Rieder, Stephanie E.; Emr, Scott D.; Jones, Elizabeth W.

doi:10.1091/mbc.7.4.579

Cited by 273 publications

(335 citation statements)

References 123 publications

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“…Proteins associated with larger organelles such as mitochondria, the vacuole, the ER, or the plasma membrane are mostly found in the P13 pellet, whereas proteins associated with the Golgi or with smaller vesicles are mostly found in the P100 pellet. Typically, the endosomal marker Pep12 is evenly distributed among the two pellet fractions (Becherer et al, 1996). This experiment, therefore, suggests that mUbp1 is localized to a larger intracellular compartment and argues against an endosomal or Golgi localization of mUbp1.…”

Section: Ubp1 Exists In Two Forms That Can Be Separated By Cell Fractmentioning

confidence: 73%

The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

Schmitz

Kinner

Kölling

2005

MBoC

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Deubiquitinating enzymes (Dubs) are potential regulators of ubiquitination-dependent processes. Here, we focus on a member of the yeast ubiquitin-specific processing protease (Ubp) family, the Ubp1 protein. We could show that Ubp1 exists in two forms: a longer membrane-anchored form (mUbp1) and a shorter soluble form (sUbp1) that seem to be independently expressed from the same gene. The membrane-associated mUbp1 variant could be localized to the endoplasmic reticulum (ER) membrane by sucrose density gradient centrifugation and by immunofluorescence microscopy. Overexpression of the soluble Ubp1 variant stabilizes the ATP-binding cassette-transporter Ste6, which is transported to the lysosome-like vacuole for degradation, and whose transport is regulated by ubiquitination. Ste6 stabilization was not the result of a general increase in deubiquitination activity, because overexpression of Ubp1 had no effect on the degradation of the ER-associated degradation substrate carboxypeptidase Y* and most importantly on Ste6 ubiquitination itself. Also, overexpression of another yeast Dub, Ubp3, had no effect on Ste6 turnover. This suggests that the Ubp1 target is a component of the protein transport machinery. On Ubp1 overexpression, Ste6 accumulates at the cell surface, which is consistent with a role of Ubp1 at the internalization step of endocytosis or with enhanced recycling to the cell surface from an internal compartment. INTRODUCTIONMany cellular proteins are modified by the attachment of the 76-amino acid polypeptide ubiquitin (Hochstrasser, 1996;Hershko and Ciechanover, 1998). The main role of ubiquitination is to target proteins for degradation either directly by acting as a degradation signal that is recognized by the 26S proteasome or indirectly by sorting membrane proteins into the lysosomal/vacuolar degradation pathway (Hicke, 1999). Ubiquitination of substrate proteins, which occurs by a cascade of enzymatic reactions, is reversible. Ubiquitin can again be removed from proteins by deubiquitinating enzymes (Dubs). A large number of Dubs have been identified in various organisms that are either cysteine proteases or metalloproteases (Verma et al., 2002; Yao and Cohen, 2002). The more classical cysteine proteases can again be divided into two groups: the ubiquitin C-terminal hydrolases (Uchs) that preferentially cleave ubiquitin from peptides and small adducts (e.g., Yuh1 in yeast) and the extremely divergent family of ubiquitin-specific processing proteases (Ubps) that cleave ubiquitin from protein substrates (Hochstrasser, 1996). Of the 17 cysteine-protease-type Dubs in yeast 16 belong to the Ubp class.The degree of ubiquitination seems to be mainly regulated at the level of ubiquitin attachment. However, evidence is accumulating that deubiquitination also can be important for the regulation of ubiquitination levels. Evidence has been presented that the deubiquitinating enzyme Fat facets (Faf), which is involved in eye development in Drosophila, acts as a substrate-specific regulator of ubiquitination of the...

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Section: Ubp1 Exists In Two Forms That Can Be Separated By Cell Fractmentioning

confidence: 73%

The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

Schmitz

Kinner

Kölling

2005

MBoC

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“…An amino acid sequence database search revealed a region of 20% identity over a 108 amino acid residue stretch between ARAP1 (residues 95 to 202) and yeast PEP12, without any gaps. The yeast PEP12 protein was localized in the pre-vacuolar endosome and its activity is required for transporting proteins from the Golgi to the vacuoles [109], [110]. This suggests the possible role of ARAP1 in intracellular traffick- The angiotensin II type 1 receptor and receptor-associated proteins ing of the AT1 receptor.…”

Section: Identification Of At1 Receptor-associated Proteinsmentioning

confidence: 98%

The angiotensin II type 1 receptor and receptor-associated proteins

et al. 2001

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The mechanisms of regulation, activation and signal transduction of the angiotensin II (Ang II) type 1 (AT1) receptor have been studied extensively in the decade after its cloning. The AT1 receptor is a major component of the renin-angiotensin system (RAS). It mediates the classical biological actions of Ang II. Among the structures required for regulation and activation of the receptor, its carboxyl-terminal region plays crucial roles in receptor internalization, desensitization and phosphorylation. The mechanisms involved in heterotrimeric G-protein coupling to the receptor, activation of the downstream signaling pathway by G proteins and the Ang II signal transduction pathways leading to specific cellular responses are discussed. In addition, recent work on the identification and characterization of novel proteins associated with carboxyl-terminus of the AT1 receptor is presented. These novel proteins will advance our understanding of how the receptor is internalized and recycled as they provide molecular mechanisms for the activation and regulation of G-protein-coupled receptors.

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“…Yeast offers an exceptional opportunity to investigate this issue because its genome contains only seven t-SNAREs (Pelham, 2001a). Thus, our gradient fractions were also probed with antibodies recognizing the t-SNAREs of the late endosome (Pep12), cis-Golgi compartment (Sed5), vacuole (Vam3), and plasma membrane (Sso1 and Sso2) (Aalto et al, 1993;Banfield et al, 1994;Becherer et al, 1996;Burd et al, 1997;Nichols et al, 1997). None of these proteins displayed complete cofractionation with the PAS marker Atg9 ( Figure 2E).…”

Section: Molecular Biology Of the Cell 2192mentioning

confidence: 99%

Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae

Reggiori

Wang

Nair

et al. 2004

MBoC

129

163

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The Cvt pathway is a biosynthetic transport route for a distinct subset of resident yeast vacuolar hydrolases, whereas macroautophagy is a nonspecific degradative mechanism that allows cell survival during starvation. Yet, these two vacuolar trafficking pathways share a number of identical molecular components and are morphologically very similar. For example, one of the hallmarks of both pathways is the formation of double-membrane cytosolic vesicles that sequester cargo before vacuolar delivery. The origin of the vesicle membrane has been controversial and various lines of evidence have implicated essentially all compartments of the endomembrane system. Despite the analogies between the Cvt pathway and autophagy, earlier work has suggested that the origin of the engulfing vesicle membranes is different; the endoplasmic reticulum is proposed to be required only for autophagy. In contrast, in this study we demonstrate that the endoplasmic reticulum and/or Golgi complex, but not endosomal compartments, play an important role for both yeast transport routes. Along these lines, we demonstrate that Berkeley bodies, a structure generated from the Golgi complex in sec7 cells, are immunolabeled with Atg8, a structural component of autophagosomes. Finally, we also show that none of the yeast t-SNAREs are located at the preautophagosomal structure, the presumed site of double-membrane vesicle formation. Based on our results, we propose two models for Cvt vesicle biogenesis. INTRODUCTIONThe lysosome/vacuole is the major cellular center for degradation, recycling, and storage of biological constituents. Several well-characterized transport routes are implicated in protein delivery to this organelle during normal growth conditions. These pathways are conserved among eukaryotic organisms, but work in the yeast Saccharomyces cerevisiae has had a major impact on their characterization and in the identification of the molecular machinery (Conibear and Stevens, 1998). The route called the alkaline phosphatase pathway provides a direct transport connection between the Golgi complex and the vacuole Piper et al., 1997). A second itinerary from the Golgi complex to the vacuole passes through the late endosome and is known as the carboxypeptidase Y pathway (Piper et al., 1995;Babst et al., 1998;Conibear and Stevens, 1998). These two pathways are required for the biogenesis and maintenance of vacuoles, whereas a third route, endocytosis, has a catabolic function. Endocytosis mediates the downregulation of plasma membrane proteins by delivering them via the late endosome to the vacuole for degradation (Hicke, 1999;Katzmann et al., 2002). Before reaching the late endosome, these proteins pass through the early endosome where some components are recycled back to the cell surface (Hicke et al., 1997;Prescianotto-Baschong and Riezman, 1998;Lewis et al., 2000). The carboxypeptidase Y pathway and endocytosis converge at the endosome where another route, the multivesicular body pathway, delivers both resident enzymes and substrates to the vac...

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Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast.

Cited by 273 publications

References 123 publications

The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

The Deubiquitinating Enzyme Ubp1 Affects Sorting of the ATP-binding Cassette-Transporter Ste6 in the Endocytic Pathway

The angiotensin II type 1 receptor and receptor-associated proteins

Early Stages of the Secretory Pathway, but Not Endosomes, Are Required for Cvt Vesicle and Autophagosome Assembly inSaccharomyces cerevisiae

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