A new type of coated vesicular carrier that appears not to contain clathrin: Its possible role in protein transport within the Golgi stack

doi:10.1016/0092-8674(86)90734-8

Cell

Volume 46, Issue 2, 18 July 1986, Pages 171-184

https://doi.org/10.1016/0092-8674(86)90734-8 Get rights and content

Abstract

Isolated Golgi membranes incubated in the presence of ATP and a cytosolic protein fraction form a population of coated buds or vesicles from the Golgi cisternae. The coats do not have the characteristic hexagonal-pentagonal basketwork of clathrin, and do not react with anti-clathrin polyclonal antibody. The conditions that produce these apparently nonclathrin-coated buds also reconstitute protein transport between compartments of the Golgi stack. The membrane of the buds contains the glycoprotein in transit through these Golgi stacks (VSV-encoded G protein). This suggests that protein transport through the Golgi stack is mediated by a new type of coated vesicle that does not contain clathrin. The concentration of G protein in the coated buds reflects the local concentration of G protein in the cisternae, raising the possibility that the Golgi coated vesicles may be “bulk” membrane carriers.

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Near the rims of the Golgi cisternae, there are many COPI-dependent vesicles with a diameter of 52 nm (Marsh et al., 2001; Mironov and Arvan, 2008; Fig. 2A: white arrows). COPI-dependent vesicles were first described by Orci et al. (1986), and these form from COPI-coated buds that are visible on the rims of the Golgi cisternae, the CMC and the ERES. The COPI coat on these buds is 10-nm thick, and it has an electron density similar to that of the external layer of the membranes (Marsh et al., 2001).
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Arf1 belongs to the Arf family of small GTPases that localise at the Golgi and plasma membrane. Active Arf1 plays a crucial role in regulating Golgi organisation and function. In mouse fibroblasts, loss of adhesion triggers a consistent drop (∼50%) in Arf1 activation that causes the Golgi to disorganise but not fragment. In suspended cells, the trans-Golgi (GalTase) disperses more prominently than cis-Golgi (Man II), accompanied by increased active Arf1 (detected using GFP-ABD: ARHGAP10 Arf1 binding domain) associated with the cis-Golgi compartment. Re-adhesion restores Arf1 activation at the trans-Golgi as it reorganises. Arf1 activation at the Golgi is regulated by Arf1 Guanine nucleotide exchange factors (GEFs), GBF1, and BIG1/2. In non-adherent fibroblasts, the cis-medial Golgi provides a unique setting to test and understand the role GEF-mediated Arf1 activation has in regulating Golgi organisation. Labelled with Man II-GFP, non-adherent fibroblasts treated with increasing concentrations of Brefeldin-A (BFA) (which inhibits BIG1/2 and GBF1) or Golgicide A (GCA) (which inhibits GBF1 only) comparably decrease active Arf1 levels. They, however, cause a concentration-dependent increase in cis-medial Golgi fragmentation and fusion with the endoplasmic reticulum (ER). Using selected BFA and GCA concentrations, we find a change in the kinetics of Arf1 inactivation could mediate this by regulating cis-medial Golgi localisation of GBF1. On loss of adhesion, a ∼50% drop in Arf1 activation over 120 min causes the Golgi to disorganise. The kinetics of this drop, when altered by BFA or GCA treatment causes a similar decline in Arf1 activation but over 10 min. This causes the Golgi to now fragment which affects cell surface glycosylation and re-adherent cell spreading. Using non-adherent fibroblasts this study reveals the kinetics of Arf1 inactivation, with active Arf1 levels, to be vital for Golgi organisation and function.
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Cell

ArticleA new type of coated vesicular carrier that appears not to contain clathrin: Its possible role in protein transport within the Golgi stack

Abstract

Cell

Cell

Cell

Cell

Cell

Cell

J. Biol. Chem.

Specimen preparation for electron microscopy using low temperature embedding resins

J. Micros.

The “coat” of kidney intercalated cell tubulovesicles does not contain clathrin

Am. J. Physiol.

Early and late functions associated with the Golgi apparatus reside in distinct compartments

Progress in unraveling pathways of Golgi traffic

Ann. Rev. Cell Biol.

Transport of vesicular stomatitis virus glycoprotein in a cell-free extract

Coated pits, coated vesicles, and receptor-mediated endocytosis

Nature

Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane

J. Cell Biol.

Article
A new type of coated vesicular carrier that appears not to contain clathrin: Its possible role in protein transport within the Golgi stack