Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport

Love, Harold D.; Lin, Chung Chih; Short, Craig; Ostermann, Joachim

doi:10.1083/jcb.140.3.541

Cited by 88 publications

(117 citation statements)

References 44 publications

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“…This recycling helps to ensure that steady-state distributions of resident proteins are maintained within the pathway at the same time as keeping a lipid balance. Besides intra-Golgi recycling between cisternae (Hoe et al, 1995;Love et al, 1998;Lanoix et al, 1999;Lin et al, 1999), Golgi glycosylation enzymes also can recycle directly to the endoplasmic reticulum (ER). It is estimated that at least 5% of Golgi resident enzymes reside in the ER, at steady state (Pelletier et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

Young

Stauber

Nery

et al. 2005

MBoC

104

126

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The small GTPase rab6A but not the isoform rab6A has previously been identified as a regulator of the COPIindependent recycling route that carries Golgi-resident proteins and certain toxins from the Golgi to the endoplasmic reticulum (ER). The isoform rab6A has been implicated in Golgi-to-endosomal recycling. Because rab6A but not A , binds rabkinesin6, this motor protein is proposed to mediate COPI-independent recycling. We show here that both rab6A and rab6A GTP-restricted mutants promote, with similar efficiency, a microtubule-dependent recycling of Golgi resident glycosylation enzymes upon overexpression. Moreover, we used small interfering RNA mediated down-regulation of rab6A and A' expression and found that reduced levels of rab6 perturbs organization of the Golgi apparatus and delays Golgi-to-ER recycling. Rab6-directed Golgi-to-ER recycling seems to require functional dynactin, as overexpression of p50/dynamitin, or a C-terminal fragment of Bicaudal-D, both known to interact with dynactin inhibit recycling. We further present evidence that rab6-mediated recycling seems to be initiated from the trans-Golgi network. Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins. INTRODUCTIONAnterograde flow of material through the exocytic pathway is offset by the constant recycling of both proteins and lipids. This recycling helps to ensure that steady-state distributions of resident proteins are maintained within the pathway at the same time as keeping a lipid balance. Besides intra-Golgi recycling between cisternae (Hoe et al., 1995;Love et al., 1998;Lanoix et al., 1999;Lin et al., 1999), Golgi glycosylation enzymes also can recycle directly to the endoplasmic reticulum (ER). It is estimated that at least 5% of Golgi resident enzymes reside in the ER, at steady state (Pelletier et al., 2000). This pool constitutes recycled material originating from the Golgi apparatus (Cole et al., 1998;Storrie et al., 1998).The extent of ER recycling of Golgi resident membrane proteins is sufficient to allow for complete assembly of functional Golgi stacks. Thus, upon addition of nocodazole to depolymerize microtubules, the central and juxtanuclear Golgi apparatus relocates to the 100 or so peripheral ER exit sites scattered throughout the cell. This microtubule-independent relocation occurs in the absence of any detectable elements tracking outwards from the central Golgi apparatus. Rather, the Golgi apparatus undergoes a molecular deconstruction in the trans-to-cis-direction, i.e., resident glycosylation enzymes of the trans-cisternae/trans-Golgi network (TGN) relocate with faster kinetics than do enzymes of the medial-cisternae (Yang and Storrie, 1998). Enzymes seemingly enter the ER close to the microtubule organizing center (MTOC), diffuse through the ER, and then reemerge at peripheral ER exit sites. Here, Golgi stacks are then reassembled into discrete, yet fully functiona...

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Section: Introductionmentioning

confidence: 99%

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

Young

Stauber

Nery

et al. 2005

MBoC

104

126

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“…Percolating COPI vesicles (Fig. 5) containing even low levels of glycosyltransferases, along with anterograde-targeted cargo, would also explain why cell-free incubations of Golgi membranes result both in transfer of VSV G protein from glycosyltransferase-free ''donor'' stacks to glycosyltransferase-containing ''acceptor'' stacks (19 -21, 49), and in transfer of catalytically active glycosyltransferase from ''acceptor'' stacks to ''donor'' stacks (50). In the hindsight of two decades, the cell-free Golgi transport assay (49) can be seen to reconstitute a spectrum of activities of COPI-coated vesicles, ref lecting a then-unanticipated richness of their role in establishing the pattern of macromolecular f low within the Golgi stack.…”

Section: Gos 28 Resides In the Same Copi-coated Buds And Vesicles Asmentioning

confidence: 99%

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

Orci

Ravazzola

Volchuk

et al. 2000

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

103

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How do secretory proteins and other cargo targeted to post-Golgi locations traverse the Golgi stack? We report immunoelectron microscopy experiments establishing that a Golgi-restricted SNARE, GOS 28, is present in the same population of COPI vesicles as anterograde cargo marked by vesicular stomatitis virus glycoprotein, but is excluded from the COPI vesicles containing retrograde-targeted cargo (marked by KDEL receptor). We also report that GOS 28 and its partnering t-SNARE heavy chain, syntaxin 5, reside together in every cisterna of the stack. Taken together, these data raise the possibility that the anterograde cargo-laden COPI vesicles, retained locally by means of tethers, are inherently capable of fusing with neighboring cisternae on either side. If so, quanta of exported proteins would transit the stack in GOS 28 -COPI vesicles via a bidirectional random walk, entering at the cis face and leaving at the trans face and percolating up and down the stack in between. Percolating vesicles carrying both post-Golgi cargo and Golgi residents up and down the stack would reconcile disparate observations on Golgi transport in cells and in cell-free systems.

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“…Previously it was hypothesized that cycling vesicles containing Golgi components fuse with ERGIC membranes (Love et al, 1998;Lin et al, 1999;Marra et al, 2001;Puthenveedu and Linstedt, 2001), therefore we wished to test whether the peripheral GalNAcT2-GFP objects were positive for ERGIC markers. However, the ERGIC collapses in the absence of ER exit (Aridor and Balch, 2000;Lee and Linstedt, 2000).…”

Section: Cycling Is Through the Er-golgi Intermediate Compartmentmentioning

confidence: 99%

Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery

Jarvela

Linstedt

2012

Journal of Cell Science

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SummaryAcute inhibition is a powerful technique to test proteins for direct roles and order their activities in a pathway, but as a general genebased strategy, it is mostly unavailable in mammalian systems. As a consequence, the precise roles of proteins in membrane trafficking have been difficult to assess in vivo. Here we used a strategy based on a genetically encoded fluorescent protein that generates highly localized and damaging reactive oxygen species to rapidly inactivate exit from the endoplasmic reticulum (ER) during live-cell imaging and address the long-standing question of whether the integrity of the Golgi complex depends on constant input from the ER. Lightinduced blockade of ER exit immediately perturbed Golgi membranes, and surprisingly, revealed that cis-Golgi-resident proteins continuously cycle to peripheral ER-Golgi intermediate compartment (ERGIC) membranes and depend on ER exit for their return to the Golgi. These experiments demonstrate that ER exit and extensive cycling of cis-Golgi components to the cell periphery sustain the mammalian Golgi complex.

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Isolation of Functional Golgi-derived Vesicles with a Possible Role in Retrograde Transport

Cited by 88 publications

References 44 publications

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

Regulation of Microtubule-dependent Recycling at theTrans-Golgi Network by Rab6A and Rab6A'

Anterograde flow of cargo across the Golgi stack potentially mediated via bidirectional “percolating” COPI vesicles

Irradiation-induced protein inactivation reveals Golgi enzyme cycling to cell periphery

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