Fig 1: Summary of vesicle capture by golgins. a Summary of the vesicle capture activities of the six indicated golgins. Golgin-97 and golgin-245 seem likely to capture the same type of vesicle, whilst the region of GCC88 that has capture activity has a very different sequence and therefore either captures the same vesicle by a different mechanism, or a different type of vesicle with overlapping cargo. The remaining three golgins capture intra-Golgi vesicles, and it appears that those bound to TMF have a similar but not identical set of cargo to those captured by golgin-84. There appear to be two different types of vesicle captured by GMAP-210. The precise number of classes of intra-Golgi vesicle, however, remains unclear. In all cases, the vesicle is captured by an N-terminal motif, with TMF also having a capture activity in its coiled-coil region. b Some of the possible models for how a vesicle captured at the N-terminus of a golgin could move closer to the Golgi membrane so as to allow vesicle fusion. The Rab binding sites on the golgin could be used in various ways to hold the golgin N-terminus closer to the membrane, or to induce a conformational change in the golgin. Alternatively, the vesicle could be captured by a further, more distant, golgin, or by different, shorter golgins (blue), to hold the vesicle closer to the target membrane. Of course, all these models are speculative, and other models could also apply
Fig 2: Mapping the vesicle capturing activity of TMF. a Schematic diagram of human TMF with plots for the predicted degree of coiled-coil and disorder. In the mitochondrial form, the Golgi-targeting transmembrane domain (TMD) is replaced with a hemagglutinin (HA) tag (h) and the TMD of human monoamine oxidase A (m). b Summary of the vesicle capture activity of the indicated variants of mitochondrial TMF. Capture at mitochondria was assayed by immunofluorescent staining of the Golgi integral membrane proteins golgin-84, giantin, and GalNAc-T2. Plus sign indicates that capture of all three markers was similar to the wild-type protein, minus sign indicates that no significant capture was observed. c Confocal micrographs of HeLa cells expressing the indicated TMF variants and stained for the HA tag on the golgin-84 chimera as well as for giantin that is in vesicles captured by TMF and for golgin-245, a protein that remains Golgi associated. Cells were treated with nocodazole for 6 h prior to fixation to ensure that mitochondria were close to intra-Golgi transport vesicles. Key constructs from the set shown in (b) are included, with similar results obtained using the markers golgin-84 and GalNAc-T2. Scale bars 10 μm. d Alignment of the N-terminus of human TMF with that from the indicated species. Bird, G. gallus; frog, X. tropicalis; fish D. rerio; urchin, S. purpuratus; bee, A. mellifera; oyster, C. gigas; hydra, H. vulgaris; sponge, A. queenslandica
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