An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes

Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/1994; 269(5):3135-8.
Source: PubMed


The Ras-related protein ADP-ribosylation factor 1 (ARF1) is a low molecular weight GTP binding protein, which in its GTP state supports the binding of coatomer, a cytosolic coat protein complex, to Golgi membranes. To create an "active" ARF, we constructed a point mutation in ARF1, Q71I, which was predicted to slow the rate of GTP hydrolysis. We demonstrate that Q71I, in contrast to wild type ARF1, exhibits a 2-3-fold increase in the half-life of ARF-GTP and is able to promote stable coatomer binding to Golgi membranes in the presence of GTP in vitro. Additionally, Q71I is able to support the binding of a significant amount of coatomer to membranes in the absence of added nucleotides, effectively bypassing the brefeldin A (BFA)-sensitive exchange activity. Furthermore, transfection of cells with Q71I, but not ARF1, renders the Golgi association of coatomer resistant to the effects of BFA in vivo. These observations provide compelling evidence that ARF1 is a necessary GTP binding protein that regulates the reversible binding of coat proteins to Golgi membranes and that the effects of BFA on this process in living cells must be a consequence of BFA's inhibition of guanine nucleotide exchange onto ARF1.

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    • "The membrane dissociation of YFP-GDAP1 induced by BFA (Fig. 3) suggests that the association of GDAP1 Figure 2. Coexpression of active ARF1 and GDAP1 enhances the recruitment of the golgin onto the Golgi apparatus. A, Confocal images of the YFP channel only of cells expressing either YFP-GDAP1 alone or YFP-GDAP1 with untagged ARF1 Q71L , which mimics the active form of ARF1 (Teal et al., 1994; Pimpl et al., 2000). The presence of untagged ARF1 in cells was ensured by using a double cistronic vector encoding ARF1 Q71L and the peroxisomal marker, CFP-SKL (Stefano et al., 2006a). "
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    ABSTRACT: Recent evidence indicates that ADP-ribosylation factor 1 (ARF1) carries out multiple roles in plant cells that may be independent from the established effector complex COPI. To investigate potential COPI-independent functions, we have followed the dynamics of ARF1 and a novel putative effector, the plant golgin GRIP-related ARF-binding domain-containing Arabidopsis (Arabidopsis thaliana) protein 1 (GDAP1) in living plant cells. We present data that ascribe a new role to ARF1 in plant cell membrane traffic by showing that the GTPase functions to recruit GDAP1 to membranes. In addition, although ARF1 appears to be central to the recruitment of both COPI components and the golgin, we have established a different subcellular distribution of these ARF1 effectors. Live cell imaging demonstrates that GDAP1 and COPI are distributed on Golgi membranes. However, GDAP1 is also found on ARF1-labeled structures that lack coatomer, suggesting that the membrane environment, rather than ARF1 alone, influences the differential recruitment of ARF1 effectors. In support of this hypothesis, fluorescence recovery after photobleaching analyses demonstrated that GDAP1 and COPI have different kinetics on membranes during the cycle of activation and inactivation of ARF1. Therefore, our data support a model where modulation of the cellular functions of ARF1 in plant cells encompasses not only the intrinsic activities of the effectors, but also differential recruitment onto membranes that is spatially regulated.
    Plant physiology 05/2007; 143(4):1615-27. DOI:10.1104/pp.106.094953 · 6.84 Impact Factor
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    • "ched the fluorescence of Arf1GTP - GFP on Golgi stacks . Upon photobleaching Arf1GTP - GFP , the fluores - cence recovered with a half - time of 35 . 7 AE 3 . 4 sec ( n ¼ 11 ) , which is threefold slower than the Arf1 - GFP half - time ( 12 . 1 AE 2 . 1 sec , n ¼ 12 , see Figure 3a , b ) in accordance with results in mammalian cells and in vitro ( Teal et al . , 1994 ; Vasudevan et al . , 1998 ) . Taken together , our results indicate that inactive Arf1 prevents COPI formation at the Golgi apparatus while the active Arf1 mutant allows COPI formation and COPI - medi - ated protein transport with a reduced activity in comparison with wild - type Arf1 ."
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    ABSTRACT: Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.
    The Plant Journal 05/2006; 46(1):95-110. DOI:10.1111/j.1365-313X.2006.02675.x · 5.97 Impact Factor
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    • "As a result, coat proteins recruited by ARF1 are translocated from the cytosol to the membrane (Donaldson et al., 1992; Tsai et al., 1992; Palmer et al., 1993). Subsequently, hydrolysis of its bound GTP deactivates ARF1, followed by release of ARF1 and coat proteins from the membrane to the cytosol (Tanigawa et al., 1993; Teal et al., 1994). "
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    ABSTRACT: ADP-ribosylation factors (ARFs) are ubiquitous regulators of virtually every step of vesicular membrane traffic. Yeast Arf3p, which is most similar to mammalian ARF6, is not essential for cell viability and not required for endoplasmic reticulum-to-Golgi protein transport. Although mammalian ARF6 has been implicated in the regulation of early endocytic transport, we found that Arf3p was not required for fluid-phase, membrane internalization, or mating-type receptor-mediated endocytosis. Arf3p was partially localized to the cell periphery, but was not detected on endocytic structures. The nucleotide-binding, N-terminal region, and N-terminal myristate of Arf3p are important for its proper localization. C-Terminally green fluorescent protein-tagged Arf3, expressed from the endogenous promoter, exhibited a polarized localization to the cell periphery and buds, in a cell cycle-dependent manner. Arf3-GFP achieved its proper localization during polarity growth through an actin-independent pathway. Both haploid and homologous diploid arf3 mutants exhibit a random budding defect, and the overexpression of the GTP-bound form Arf3p(Q71L) or GDP-binding defective Arf3p(T31N) mutant interfered with budding-site selection. We conclude that the GTPase cycle of Arf3p is likely to be important for the function of Arf3p in polarizing growth of the emerging bud and/or an unidentified vesicular trafficking pathway.
    Molecular Biology of the Cell 10/2003; 14(9):3834-47. DOI:10.1091/mbc.E03-01-0013 · 4.47 Impact Factor
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