Conformational changes of coat proteins during vesicle formation

Langer, Julian D.; Stoops, Emily H.; Béthune, Julien; Wieland, Felix

doi:10.1016/j.febslet.2007.03.020

Cited by 18 publications

(17 citation statements)

References 75 publications

(92 reference statements)

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“…Based on these results, we hypothesize that cargo affects GAP activity by changing the conformation of coatomer to a form that more efficiently binds to and/or activates Arf GAP2. Consistent with this idea, cargo has been previously reported to affect the conformation of coatomer [44,45] and of clathrin adaptor protein-1 [46]. Cargo peptide also stimulated Arf GAP1 when coatomer was present leading us to postulate that the effect of cargo we have described for Arf GAP2 and coatomer may extend to other Arf GAP/coat pairs.…”

Section: Discussionsupporting

confidence: 80%

Arf GAP2 is positively regulated by coatomer and cargo

Luo¹,

Ha²,

Hayashi³

et al. 2009

Cellular Signalling

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Arf GAP2 is one of four Arf GAPs that function in the Golgi apparatus. We characterized the kinetics of Arf GAP2 and its regulation. Purified Arf GAP2 had little activity compared to purified Arf GAP1. Of the potential regulators we examined, coatomer had the greatest effect, stimulating activity one to two orders of magnitude. The effect was biphasic, with half-maximal activation observed at 50 nM coatomer and activation peaking at ≈150 nM coatomer. Activation by coatomer was greater for Arf GAP2 than has been reported for Arf GAP1. The effects of phosphoinositides and changes in vesicle curvature on GAP activity were small compared to coatomer; however, both increased coatomer-dependent activity. Peptides from p24 cargo proteins increased Arf GAP2 activity by an additional 2-to 4-fold. The effect of cargo peptide was dependent on coatomer. Overexpressing the cargo protein p25 decreased cellular Arf1•GTP levels. The differential sensitivity of Arf GAP1 and Arf GAP2 to coatomer could coordinate their activities. Based on the common regulatory features of Arf GAP1 and 2, we propose a mechanism for cargo selection in which GTP hydrolysis triggered by cargo binding to the coat protein is coupled to coat polymerization.

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Section: Discussionsupporting

confidence: 80%

Arf GAP2 is positively regulated by coatomer and cargo

Luo¹,

Ha²,

Hayashi³

et al. 2009

Cellular Signalling

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“…Currently, it is not clear if ‘spiky coats’ and ‘homogeneous coats’ represent distinct subtypes differing in genesis and protein composition, or if they are COPI coats at different stages of coating/uncoating. Strong evidence for functionally different COPI vesicles comes from biochemical (Malsam et al , 2005) and recent immuno‐EM data (Langer et al , 2007), reviewed in (Bethune et al , 2006). But it is unclear, if slight composition differences would imply such structural diversification as we observed.…”

Section: Discussionmentioning

confidence: 99%

Golgi apparatus studied in vitreous sections

Bouchet‐Marquis

Starkuviene

Grabenbauer

2008

Journal of Microscopy

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SummaryCryo-electron microscopy of vitrified specimen is the method of choice to explore cellular ultrastructure at high resolution as close as possible to the native state and environment. In this study, we investigated the Golgi apparatus -the main organelle of the secretory pathway. Cultured mammalian cells were fixed by high-pressure freezing, sectioned in vitreous ice and subjected to cryo-electron microscopy and cryo-electron tomography. Although the overall morphology of Golgi stacks was comparable to well prepared and plastic-embedded samples, in detail we reached much higher resolution in terms of distinction between biological structures based on their native density. On cisternal buds and peri-Golgi vesiclessome associated with microtubules -we detected two different subtypes of COPI coats: (1) a homogenous coat and (2) an inhomogeneous spiky coat, providing an 8-9 nm regularity, clearly distinct from clathrin coat. Next, we monitored the secretion of cargo, namely, procollagen I, through the Golgi complex. Temporally correlated with fluorescence microscopy, we performed three-dimensional cryo-electron tomography analysis and detected Golgi cisternae enlarged to saccules, containing cargo and showing inter-cisternal connections. Our work provides a first step towards the high-resolution description of the secretory pathway in native vitrified samples and describes the challenges associated with this attempt.

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“…Protein phosphorylation modulates multiple events along the endocytic and exocytic pathways: coat polymerization, membrane tethering, and SNARE assembly (Langer et al, 2007;Preisinger and Barr, 2005). Here, we have analyzed how phosphorylation of Vps41 can control its function in the tethering stage at the endosome/vacuole interface.…”

Section: Discussionmentioning

confidence: 99%

Vps41 Phosphorylation and the Rab Ypt7 Control the Targeting of the HOPS Complex to Endosome–Vacuole Fusion Sites

Cabrera

Ostrowicz

Mari

et al. 2009

MBoC

139

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Membrane fusion depends on multisubunit tethering factors such as the vacuolar HOPS complex. We previously showed that the vacuolar casein kinase Yck3 regulates vacuole biogenesis via phosphorylation of the HOPS subunit Vps41. Here, we link the identified Vps41 phosphorylation site to HOPS function at the endosome-vacuole fusion site. The nonphosphorylated Vps41 mutant (Vps41 S-A) accumulates together with other HOPS subunits on punctate structures proximal to the vacuole that expand in a class E mutant background and that correspond to in vivo fusion sites. Ultrastructural analysis of this mutant confirmed the presence of tubular endosomal structures close to the vacuole. In contrast, Vps41 with a phosphomimetic mutation (Vps41 S-D) is mislocalized and leads to multilobed vacuoles, indicative of a fusion defect. These two phenotypes can be rescued by overproduction of the vacuolar Rab Ypt7, revealing that both Ypt7 and Yck3-mediated phosphorylation modulate the Vps41 localization to the endosome-vacuole junction. Our data suggest that Vps41 phosphorylation fine-tunes the organization of vacuole fusion sites and provide evidence for a fusion "hot spot" on the vacuole limiting membrane.

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Conformational changes of coat proteins during vesicle formation

Cited by 18 publications

References 75 publications

Arf GAP2 is positively regulated by coatomer and cargo

Arf GAP2 is positively regulated by coatomer and cargo

Golgi apparatus studied in vitreous sections

Vps41 Phosphorylation and the Rab Ypt7 Control the Targeting of the HOPS Complex to Endosome–Vacuole Fusion Sites

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