Modulation of the Golgi apparatus in <i>Saccharomyces cerevisiae sec7</i> mutants as seen by three‐dimensional electron microscopy

Rambourg, A.; Clermont, Y.; Képès, François

doi:10.1002/ar.1092370402

Cited by 71 publications

(83 citation statements)

References 23 publications

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“…Both are large double-membrane vesicles enwrapping ribosomes and cytosol (Novick et al, 1980;Baba et al, 1994;Figure 6). We have now shown that Atg8 localizes to membranes derived from the Golgi complex and/or ER that give rise to Berkeley bodies (Novick et al, 1980;Esmon et al, 1981;Rambourg et al, 1993;Rambourg et al, 1996;Figure 6).…”

Section: The Pas and Double-membrane Vesicle Biogenesis Depends On Eamentioning

confidence: 94%

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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Section: The Pas and Double-membrane Vesicle Biogenesis Depends On Eamentioning

confidence: 94%

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 Golgi stacks were located next to tER sites and exhibited a distinct polarity due to a differential staining of the lumenal contents of the cisternae by OsO 4 . Most likely, these differences in staining reflect a progressively heavier glycosylation of glycoproteins during passage through the Golgi (Zhang and Staehelin, 1992;Rambourg et al, 1993;Munro, 2001). Individual vesicles were observed throughout the cytoplasm, and secretory granules were clustered under emerging buds at the plasma membrane.…”

Section: Cryofixation Of P Pastoris Cells Yields Excellent Preservatmentioning

confidence: 99%

Tomographic Evidence for Continuous Turnover of Golgi Cisternae inPichia pastoris

Møgelsvang

Gomez‐Ospina

Soderholm

et al. 2003

MBoC

132

141

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The budding yeast Pichia pastoris contains ordered Golgi stacks next to discrete transitional endoplasmic reticulum (tER) sites, making this organism ideal for structure-function studies of the secretory pathway. Here, we have used P. pastoris to test various models for Golgi trafficking.The experimental approach was to analyze P. pastoris tER-Golgi units by using cryofixed and freeze-substituted cells for electron microscope tomography, immunoelectron microscopy, and serial thin section analysis of entire cells. We find that tER sites and the adjacent Golgi stacks are enclosed in a ribosome-excluding "matrix." Each stack contains three to four cisternae, which can be classified as cis, medial, trans, or trans-Golgi network (TGN). No membrane continuities between compartments were detected. This work provides three major new insights. First, two types of transport vesicles accumulate at the tER-Golgi interface. Morphological analysis indicates that the center of the tER-Golgi interface contains COPII vesicles, whereas the periphery contains COPI vesicles. Second, fenestrae are absent from cis cisternae, but are present in medial through TGN cisternae. The number and distribution of the fenestrae suggest that they form at the edges of the medial cisternae and then migrate inward. Third, intact TGN cisternae apparently peel off from the Golgi stacks and persist for some time in the cytosol, and these "free-floating" TGN cisternae produce clathrin-coated vesicles. These observations are most readily explained by assuming that Golgi cisternae form at the cis face of the stack, progressively mature, and ultimately dissociate from the trans face of the stack. INTRODUCTIONThe Golgi apparatus consists of flattened membrane cisternae that are usually organized into stacks (Berger and Roth, 1997). Newly synthesized biosynthetic cargo molecules exit the endoplasmic reticulum (ER) in COPII-coated vesicles and enter the cis cisterna of the Golgi (Farquhar and Hauri, 1997;Barlowe, 2002). These cargo molecules then occur in medial and trans-cisternae. In the trans-Golgi network (TGN), the cargo molecules are sorted into different types of carriers for delivery to the plasma membrane and other destinations (Mellman and Simons, 1992). Although this basic scheme is well established, the pathway of intra-Golgi transport is still being investigated. Anterograde Golgi transport has been proposed to occur via vesicular intermediates, membrane continuities, cisternal maturation, or a combination of these mechanisms (Beznoussenko and Mironov, 2002). The extent to which a given mechanism operates may vary with the cell type and the stage of the cell cycle (Pelham and Rothman, 2000;Marsh and Howell, 2002). Considerable evidence now favors cisternal maturation as a major route for intra-Golgi transport (Pelham, 2001;Storrie and Nilsson, 2002), but the generality and several key predictions of this model remain to be verified.We are using electron microscopy to test predictions of the different Golgi-trafficking models. Because some of the...

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“…This phenotype is reminiscent of that of sec7 (Novick et al, 1981;Rambourg et al, 1993), a mutant of a guanine nucleotide exchange factor (GEF) of Arf (see below), but its meaning is unknown.…”

Section: Group 2 (Arf1mentioning

confidence: 99%

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

Yahara

Ueda

Sato

et al. 2001

MBoC

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ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.

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Modulation of the Golgi apparatus in Saccharomyces cerevisiae sec7 mutants as seen by three‐dimensional electron microscopy

Cited by 71 publications

References 23 publications

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

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

Tomographic Evidence for Continuous Turnover of Golgi Cisternae inPichia pastoris

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

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