Abstract
Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.
Highlights
In the secretory pathway, proteins and lipids are transported between organelles by vesicles
We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1
We found that ARFGAP2 and ARFGAP3 co-localized with coatomer on the Golgi membrane, including some of the peripheral sites labeled with anti-coatomer antibodies
Summary
Reagents—BFA, propranolol, NaF, CaCl, MgCl, NaN3, NH4Cl, formaldehyde, puromycin, saponin, fish skin gelatin, and 1,4-diazabicyclo[2.2.2]octane were from Sigma-Aldrich. Fluorescence loss in photobleaching (FLIP) was performed by taking time series of images during ϳ10 min, monitoring the fluorescence loss in the Golgi apparatus due to bleaching of the cytosol pool of fluorescent molecules. Each image in the FLIP time series was followed by a bleach event, in which the whole cell apart from the Golgi area was bleached by intense 488-nm excitation (100% transmission). The FLIP analysis was performed by fitting the decaying curve with a single exponential function, I(t) ϭ I∞ ϩ I0 ϫ e(Ϫt/T), where I∞ is the final remaining fluorescence at the end of the time series, I0 is the initial fluorescence, and T is the decay time, which is related to the half-time for the decay through t1⁄2 ϭ ln 2 ϫ T. Digital images were obtained through a side-mounted MegaView III TEM CCD camera and quantified according to Ref. 40
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