Golgi Localization Determinants in ArfGAP1 and in New Tissue-specific ArfGAP1 Isoforms

Abstract

The Arf1-directed GTPase-activating protein ArfGAP1 is a Golgi-localized protein that controls the dynamics of the COPI coat of carriers that mediate transport in the endoplasmic reticulumGolgi shuttle. Previously the interaction of ArfGAP1 with the Golgi was allocated to a portion of the non-catalytic, carboxyl part of the protein, but the mechanism of this interaction has not been established. In this study we identify a short stretch in the non-catalytic part of ArfGAP1 (residues 204 -214) in which several hydrophobic residues contribute to Golgi localization. Even single alanine replacement of two of these residues (Leu-207 and Trp-211) strongly diminished Golgi localization. Mutations in the hydrophobic residues also diminished the in vitro activity of ArfGAP1 on Arf1 bound to Golgi membranes. The stretch containing the hydrophobic residues was recently shown to mediate the binding of ArfGAP1 to loosely packed lipids of highly curved liposomes (Bigay, J., Casella, J. F., Drin, G., Mesmin, B., and Antonny, B. (2005) EMBO J. 24, 2244 -2253). Whereas short fragments containing the hydrophobic stretch were not Golgi-localized, a proximal 10-residue inframe insertion that is present in new ArfGAP1 isoforms that we identified in brain and heart tissues could confer Golgi localization on these fragments. This localization was abrogated by alanine replacement of residues Phe-240 or Trp-241 of the insertion sequence but not by their replacement with leucines. Our findings indicate that ArfGAP1 interacts with the Golgi through multiple hydrophobic motifs and that alternative modes of interaction may exist in tissue-specific ArfGAP1 isoforms.Membrane traffic in the endoplasmic reticulum-Golgi shuttle is mediated by the COPI 3 and COPII trafficking systems. The COPII system mediates the initial exit from the endoplasmic reticulum, whereas subsequent transport to the Golgi apparatus and retrograde Golgi to endoplasmic reticulum traffic involves COPI carriers. The COPI and COPII coats are composed of evolutionarily distinct sets of proteins yet follow similar pathways of coat assembly and disassembly (for recent reviews see Refs. 1-3). In both systems a small GTPase (Arf1 and Sar1 for COPI and COPII, respectively) plays a key regulatory role. Following activation by a guanine nucleotide exchange protein, the GTPase translocates from cytosol to the organelle membrane, where it initiates the process of coat formation by the direct binding of coat subunits (4 -6). The coat in turn recruits cargo and polymerizes causing membrane deformation to form a bud.In the second phase of the GTPase cycle of Arf1 and Sar1, bound GTP is hydrolyzed with the aid of a GTPase-activating protein (GAP). The hydrolysis of GTP is a prerequisite for coat dissociation, and its inhibition leads to the accumulation of coated vesicles that are prevented from fusing with the target membrane (4, 7). The GAP for Sar1 is a subunit of the first layer of the COPII coat, the Sec23/24 complex (8). ArfGAPs constitute a large family of proteins containing a hi...

Arf activation at the Golgi is modulated by feed-forward stimulation of the exchange factor GBF1

“…The plasmid encoding EYFP-PH FAPP (Godi et al, 2004) was obtained through S. Grinstein (Sick Kids, ON). Construction of a plasmid encoding ArfGAP1 tagged with EGFP at the N-terminus was as previously described (Parnis et al, 2006). A point mutant lacking GAP activity (EGFP-GAP1 R50Q) was created by sitedirected mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA).…”

Topology of Amphipathic Motifs Mediating Golgi Localization in ArfGAP1 and Its Splice Isoforms

“…This motif is unstructured in solution, but upon binding to liposomes it folds into an amphipathic helix that is stabilized by the insertion of bulky hydrophobic residues into open spaces in the outer lipid bilayer. A role of the ALPS motif in vivo was demonstrated by our finding that Golgi localization of Arf-GAP1 is abrogated by point mutations of hydrophobic residues near the amino side of ALPS (19). More recently, a second amphipathic motif (ALPS2) with similar physicochemical characteristics was identified in ArfGAP1.…”

“…The cellular distribution of GFP fusions of ArfGAP1 and mutants thereof was determined as the ratio of the average pixel intensity at the Golgi to that of a thin section of cytosol in the close proximity of the Golgi. Because Golgi staining was faint in many ArfGAP1 mutants, the Golgi area was defined in all experiments by counter-staining with GM130, a cis-Golgi marker that shows a good colocalization with ArfGAP1 (19). As shown in Fig.…”

“…Localization-Previously, the role of the ALPS motifs in targeting ArfGAP1 to the Golgi was inferred from the effect of a few mutations in selective regions of these motifs (19,25,26). For a mutagenesis study aimed at defining the extent of the motifs mediating Golgi localization, it was necessary to design a quantitative analysis of ArfGAP1 interaction with the Golgi.…”

“…In addition to a ubiquitous form expressed in all rat tissues examined so far, there are tissuespecific isoforms (19). One isoform expressed in heart tissue contains an in-frame 10-residue insertion in proximity to the ALPS1 motif, whereas a second isoform that constitutes the major form of ArfGAP1 in the rat brain contained the same insertion together with a 22-residue deletion within the ALPS2 motif.…”