Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis.
(25/177)
Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus. (+info)
A role for calcium in stabilizing transport vesicle coats.
(26/177)
Calcium has been implicated in regulating vesicle fusion reactions, but its potential role in regulating other aspects of protein transport, such as vesicle assembly, is largely unexplored. We find that treating cells with the membrane-permeable calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM), leads to a dramatic redistribution of the vesicle coat protein, coatomer, in the cell. We have used the cell-free reconstitution of coat-protomer I (COPI) vesicle assembly to characterize the mechanisms of this redistribution. We find that the recovery of COPI-coated Golgi vesicles is inhibited by the addition of BAPTA to the cell-free vesicle budding assay. When coatomer-coated membranes are incubated in the presence of calcium chelators, the membranes "uncoat," indicating that calcium is necessary for maintaining the integrity of the coat. This uncoating is reversed by the addition of calcium. Interestingly, BAPTA, a calcium chelator with fast binding kinetics, is more potent at uncoating the coatomer-coated membrane than EGTA, suggesting that a calcium transient or a calcium gradient is important for stabilizing COPI vesicle coat. The primary target for the effects of calcium on coatomer recruitment is a step that occurs after ADP-ribosylation factor binding to the membrane. We suggest that a calcium gradient may serve to regulate the timing of vesicle uncoating. (+info)
Peroxisomal membrane proteins are properly targeted to peroxisomes in the absence of COPI- and COPII-mediated vesicular transport.
(27/177)
The classic model for peroxisome biogenesis states that new peroxisomes arise by the fission of pre-existing ones and that peroxisomal matrix and membrane proteins are recruited directly from the cytosol. Recent studies challenge this model and suggest that some peroxisomal membrane proteins might traffic via the endoplasmic reticulum to peroxisomes. We have studied the trafficking in human fibroblasts of three peroxisomal membrane proteins, Pex2p, Pex3p and Pex16p, all of which have been suggested to transit the endoplasmic reticulum before arriving in peroxisomes. Here, we show that targeting of these peroxisomal membrane proteins is not affected by inhibitors of COPI and COPII that block vesicle transport in the early secretory pathway. Moreover, we have obtained no evidence for the presence of these peroxisomal membrane proteins in compartments other than peroxisomes and demonstrate that COPI and COPII inhibitors do not affect peroxisome morphology or integrity. Together, these data fail to provide any evidence for a role of the endoplasmic reticulum in peroxisome biogenesis. (+info)
Evidence that Golgi structure depends on a p115 activity that is independent of the vesicle tether components giantin and GM130.
(28/177)
Inhibition of the putative coatomer protein I (COPI) vesicle tethering complex, giantin-p115-GM130, may contribute to mitotic Golgi breakdown. However, neither this, nor the role of the giantin-p115-GM130 complex in the maintenance of Golgi structure has been demonstrated in vivo. Therefore, we generated antibodies directed against the mapped binding sites in each protein of the complex and injected these into mammalian tissue culture cells. Surprisingly, the injected anti-p115 and antigiantin antibodies caused proteasome-mediated degradation of the corresponding antigens. Reduction of p115 levels below detection led to COPI-dependent Golgi fragmentation and apparent accumulation of Golgi-derived vesicles. In contrast, neither reduction of giantin below detectable levels, nor inhibition of p115 binding to GM130, had any detectable effect on Golgi structure or Golgi reassembly after cell division or brefeldin A washout. These observations indicate that inhibition of p115 can induce a mitotic-like Golgi disassembly, but its essential role in Golgi structure is independent of its Golgi-localized binding partners giantin and GM130. (+info)
KDEL-cargo regulates interactions between proteins involved in COPI vesicle traffic: measurements in living cells using FRET.
(29/177)
How the occupied KDEL receptor ERD2 is sorted into COPI vesicles for Golgi-to-ER transport is largely unknown. Here, interactions between proteins of the COPI transport machinery occurring during a "wave" of transport of a KDEL ligand were studied in living cells. FRET between CFP and YFP fusion proteins was measured by multifocal multiphoton microscopy and bulk-cell spectrofluorimetry. Ligand binding induces oligomerization of ERD2 and recruitment of ARFGAP to the Golgi, where the (ERD2)n/ARFGAP complex interacts with membrane-bound ARF1. During KDEL ligand transport, interactions of ERD2 with beta-COP and p23 decrease and the proteins segregate. Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds. (+info)
Golgi-to-endoplasmic reticulum (ER) retrograde traffic in yeast requires Dsl1p, a component of the ER target site that interacts with a COPI coat subunit.
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DSL1 was identified through its genetic interaction with SLY1, which encodes a t-SNARE-interacting protein that functions in endoplasmic reticulum (ER)-to-Golgi traffic. Conditional dsl1 mutants exhibit a block in ER-to-Golgi traffic at the restrictive temperature. Here, we show that dsl1 mutants are defective for retrograde Golgi-to-ER traffic, even under conditions where no anterograde transport block is evident. These results suggest that the primary function of Dsl1p may be in retrograde traffic, and that retrograde defects can lead to secondary defects in anterograde traffic. Dsl1p is an ER-localized peripheral membrane protein that can be extracted from the membrane in a multiprotein complex. Immunoisolation of the complex yielded Dsl1p and proteins of approximately 80 and approximately 55 kDa. The approximately 80-kDa protein has been identified as Tip20p, a protein that others have shown to exist in a tight complex with Sec20p, which is approximately 50 kDa. Both Sec20p and Tip20p function in retrograde Golgi-to-ER traffic, are ER-localized, and bind to the ER t-SNARE Ufe1p. These findings suggest that an ER-localized complex of Dsl1p, Sec20p, and Tip20p functions in retrograde traffic, perhaps upstream of a Sly1p/Ufe1p complex. Last, we show that Dsl1p interacts with the delta-subunit of the retrograde COPI coat, Ret2p, and discuss possible roles for this interaction. (+info)
Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-Golgi transport.
(31/177)
A cisternal progression mode of intra-Golgi transport requires that Golgi resident proteins recycle by peri-Golgi vesicles, whereas the alternative model of vesicular transport predicts anterograde cargo proteins to be present in such vesicles. We have used quantitative immuno-EM on NRK cells to distinguish peri-Golgi vesicles from other vesicles in the Golgi region. We found significant levels of the Golgi resident enzyme mannosidase II and the transport machinery proteins giantin, KDEL-receptor, and rBet1 in coatomer protein I-coated cisternal rims and peri-Golgi vesicles. By contrast, when cells expressed vesicular stomatitis virus protein G this anterograde marker was largely absent from the peri-Golgi vesicles. These data suggest a role of peri-Golgi vesicles in recycling of Golgi residents, rather than an important role in anterograde transport. (+info)
Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.
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BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell. (+info)