[13] Reconstitution of transport from endoplasmic reticulum to golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast

Wuestehube, Linda J.; Schekman, Randy

doi:10.1016/0076-6879(92)19015-x

Cited by 91 publications

(85 citation statements)

References 23 publications

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“…Membranes were incubated at 12°C for 15 min to stimulate translocation, 0.8 ml of B88-8 with 1% digitonin was then added and immunoprecipitations were performed as described above. The immunoprecipitations used for mass spectrometry analysis were performed from 2 A 280 units (ϳ0.4 mg of protein) of microsomes (39) and were otherwise carried out as described for semi-intact cells. Samples were resolved on a 10% Novex Tris glycine gel and stained with a Colloidal Blue Staining Kit (Invitrogen) or silver stained (40).…”

Section: Methodsmentioning

confidence: 99%

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Yet1p and Yet3p, the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

Wilson

Barlowe

2010

Journal of Biological Chemistry

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The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1⌬ and yet3⌬ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1⌬ or yet3⌬ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.

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Section: Methodsmentioning

confidence: 99%

“…In Vitro Vesicle Budding-Experiments to assess the COPIIdependent packaging of Yet1p and Yet3p were performed using microsomes (39) in 250-l reactions essentially as described (34,41).…”

Section: Methodsmentioning

confidence: 99%

Yet1p and Yet3p, the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

Wilson

Barlowe

2010

Journal of Biological Chemistry

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“…Yeast Microsome Preparations for Immunoblotting or ␥-Secretase Assay-Yeast microsomes were prepared as described previously (37). Microsomal membranes were suspended in ␥-buffer (50 mM PIPES (pH 7.0), 250 mM sucrose, 1 mM EGTA) and subjected to immunoblotting or ␥-secretase assay (38).…”

Section: Methodsmentioning

confidence: 99%

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

Futai

Yagishita

Ishiura

2009

Journal of Biological Chemistry

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␥-Secretase is a multisubunit membrane protein complex consisting of presenilin (PS1), nicastrin (NCT), anterior pharynx-1, and presenilin enhancer 2. To analyze the activity of familial Alzheimer disease mutants and to understand the roles of the subunits, we established a yeast transcriptional activator Gal4p system with artificial ␥-secretase substrates containing amyloid precursor protein or Notch fragments. The ␥-secretase activities were evaluated by transcriptional activation of reporter genes upon Gal4p release from the membrane-bound substrates, i.e. growth of yeast on histidine and adenine, or ␤-galactosidase assay. We screened and evaluated ␥-secretase mutants using this reconstitution system in yeast, which does not possess endogenous ␥-secretase activity. When we introduced familial Alzheimer mutants of PS1 in this system, their activities were shown to be loss of function. Although the protease activity of wild type PS1 depends on the other three subunits introduced, we obtained 15 new PS1 mutants, which are active in the absence of NCT. They possessed a S438P mutation at the ninth transmembrane domain (TM9) together with one missense mutation distributed through transmembrane and loop regions. These mutations were not related to familial Alzheimer mutations of PS1 as identified so far. The S438P mutant was partially active but required other mutations for full activation. Results of the ␤-galactosidase assay suggested that they have wild type protease activities, which were further confirmed by the endoproteolysis of PS1, amyloid ␤ peptides, and Notch intracellular domain production in mammalian cells. These results suggest that NCT is dispensable for the protease activity of ␥-secretase.

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“…Microsomes were prepared from strain RSY455 as described (15). In vitro vesicle-budding reactions were performed essentially as described by Barlowe et al (16).…”

Section: Methodsmentioning

confidence: 99%

Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum

Todorow

Spang

Carmack

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

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Mnn9p is a component of two distinct multiprotein complexes in the Saccharomyces cerevisiae cis-Golgi that have both been shown to have ␣-1,6-mannosyltransferase activity in vitro. In one of these complexes, Mnn9p associates with four other membrane proteins, Anp1p, Mnn10p, Mnn11p, and Hoc1p, whereas the other complex consists of Mnn9p and Van1p. Members of the Mnn9p-containing complexes were incorporated into COPII vesicles made in vitro from endoplasmic reticulum (ER) membranes isolated from cycloheximidetreated cells. This behavior is consistent with an active Golgi to ER recycling process. To examine this path in vivo, we monitored retrograde transport of subunits of the complex in cells blocked in anterograde transport from the ER. In this situation, specific relocation of the proteins from the Golgi to the ER was observed in the absence of new protein synthesis. Conversely, when retrograde transport was blocked in vivo, subunits of the mannosyltransferase complex accumulated in the vacuole. Packaging of Mnn9p in COPI-coated vesicles from purified Golgi membranes was also investigated using a coatomer-dependent vesicle budding assay. Gradient fractionation experiments showed that Mnn9p and the retrograde v-SNARE, Sec22p, were incorporated into COPI-coated vesicles. These observations indicate that the Mnn9p-containing mannosyltransferase complexes cycle back and forth between the ER and Golgi.T he biogenesis of the Golgi complex and the mechanism of intra-Golgi protein transport have long been the subject of intense interest in membrane cell biology. The origin of the different Golgi subcompartments, with their unique set of resident proteins and specialized functions, is a matter of much debate. Two models inform this debate (1, 2). The vesicular transport model posits that Golgi cisternae are stable structures that receive and create vesicles carrying cargo molecules in the anterograde direction and retrograde vesicles returning escaped proteins to their resident cisternae or the endoplasmic reticulum (ER) (3). Alternatively, in the cisternal maturation model, each Golgi cisterna translates through a stack of membranes acquiring new functional capabilities by fusing with retrograde vesicles that carry enzymes from a distal cisterna (2). According to this view, the cis-Golgi can then be considered an outgrowth of the ER, formed by the homotypic fusion of ER-derived transport vesicles. A possible resolution is that the two mechanisms operate in parallel to achieve rapid (vesicular) and slow (cisternal) transport of molecules and large particles, respectively (4).One strong piece of evidence for cisternal maturation emerged from studies in which procollagen helical fibers were tracked as they moved across the Golgi stack in collagen-secreting fibroblasts (5). Using a combination of biochemical and morphological methods, Bonfanti et al. (5) demonstrated that procollagen traverses the stack while staying enclosed in the lumen of Golgi cisternae. Independent evidence supporting this view came with the observat...

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[13] Reconstitution of transport from endoplasmic reticulum to golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast

Cited by 91 publications

References 23 publications

Yet1p and Yet3p, the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

Yet1p and Yet3p, the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

Active recycling of yeast Golgi mannosyltransferase complexes through the endoplasmic reticulum

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