Peri-Golgi vesicles contain retrograde but not anterograde proteins consistent with the cisternal progression model of intra-Golgi transport

Abstract

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.

Figure 1.

Immunogold labeling of Man II (A, B, and D, 10 nm gold; C, 15 nm gold) on ultrathin cryosections of NRK cells, showing the presence of Man II in the cis-medial Golgi cisternae (G) and in lateral rims of the cisternae and associated vesicular profiles (arrows). Note that the relative distribution of Man II between cisternae and associated vesicles varies considerably between Golgi's. The arrowhead in A points to a Man II–positive Golgi lateral rim with a clearly visible COP coat. The arrow in C points to a Man II–positive vesicle with a visible COP coat. Bars, 200 nm.

Figure 4.

VSV-G ^ts045 –GFP (10 nm gold) is present in Golgi cisternae of transfected NRK cells but devoid from lateral peri-Golgi vesicles. In A–C, the protein construct was visualized by using an antibody against the cytosolically exposed GFP tag. In D, an antibody against the luminal domain of VSV-G was used. (A) After 2.5 min at the permissive temperature, VSV-G^ts045–GFP colocalizes with KDELr (15 nm gold) in VTCs but is absent from the Golgi complex (G). KDELr is present in Golgi cisternae and in surrounding vesicles (arrows). (B) 20 min after release of the temperature block, VSV-G^ts045–GFP is readily detectable in all cisternae of the Golgi complex. Note that KDELr-positive peri-Golgi vesicles do not contain VSV-G^ts045–GFP (arrows). (C) Example of a cell with a high expression level of VSV-G^ts045–GFP. Man II (15 nm gold) distribution over the Golgi complex is not affected by VSV-G^ts045–GFP. The large arrowhead points to a clathrin-coated vesicle. (D) The antibody against the luminal part of VSV-G gives an identical staining pattern to anti-GFP with high levels in the cisternae but no label in the peri-Golgi vesicles (arrows). Bars, 200 nm.

Figure 2.

Man II (15 nm gold)–positive peri-Golgi (G) vesicles (arrows) lack COPII (10 nm gold), which is restricted to vesicles close to the ER. Bar, 200 nm.

Figure 3.

COPI (10 nm gold) colocalizes with Man II (15 nm gold) in lateral rims (arrowhead) of Golgi (G) cisternae and peri-Golgi vesicles (arrows). Bars, 100 nm.

Figure 5.

Giantin (10 nm gold) and rBet1 (15 nm gold) show different distribution patterns. Most of the cell's giantin is present in the Golgi complex (G) and associated vesicles. rBet1 labels VTC membranes and the cis-Golgi cisterna. Bar, 200 nm.

Figure 6.

Man II colocalizes with giantin and KDELr in different populations of peri-Golgi vesicles. (A) Man II (10 nm gold) colocalizes with giantin (15 nm gold) in peri-Golgi vesicles (arrow). (B) Likewise, Man II (10 nm gold) is regularly found together with KDELr (15 nm gold) in peri-Golgi vesicles (arrows). (C) Despite the high labeling densities of both KDELr (10 nm gold) and giantin (15 nm gold), they are found mostly in different populations of peri-Golgi vesicles. Arrows point to KDELr-positive and giantin-negative vesicles. The arrowhead points to a Golgi cisternal rim that is strongly labeled KDELr and lacks giantin. G, Golgi complex. Bars, 200 nm.

Figure 7.

KDELr (10 nm gold) and rBet1 (15 nm gold) colocalize in peripheral VTCs (pVTC) but not peri-Golgi vesicles. (A) High levels of KDELr and rBet1 are found in membranes of a peripheral VTC. (B) In the Golgi (G) region, rBet1 is present in irregularly shaped VTC membranes but absent from peri-Golgi vesicles (arrows). P, plasma membrane. Bars, 200 nm.