Dependence of RGS9-1 membrane attachment on its C-terminal tail.
RGS9-1 is a GTPase-accelerating protein (GAP) required for rapid recovery of the light response in vertebrate rod and cone photoreceptors. Similar to its phototransduction partners transducin (G(t)) and cGMP phosphodiesterase, it is a peripheral protein of the disc membranes, but it binds membranes much more tightly. It lacks the lipid modifications found on G(t) and cGMP phosphodiesterase, and the mechanism for membrane attachment is unknown. We have used limited proteolysis to generate a fragment of RGS9-1 that is readily removed from membranes under moderate salt conditions. Immunoblots reveal that this soluble fragment lacks a 3-kDa fragment from the C-terminal domain, the only domain within RGS9-1 that differs in sequence from the brain-specific isoform RGS9-2. Recombinant fragments of RGS9-1 with or without the partner subunit G beta(5L) were constructed with or without the C-terminal domain. Those lacking the C-terminal domain bound to photoreceptor membranes much less tightly than those containing it. Removal by urea of G beta(5L) from endogenous or recombinant RGS9-1 bound to rod outer segment membranes left RGS9-1 tightly membrane-bound, and recombinant RGS9-1 was urea-soluble in the absence of membranes. Thus the C-terminal domain of RGS9-1 is critical for membrane binding, whereas G beta(5L) does not play an important role in membrane attachment.
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Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Melia
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Cowan
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Wensel
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D000818:Animals; D002021:Buffers; D002417:Cattle; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000737:chemistry* / Q000378:metabolism; D012492:Salts / Q000737:chemistry; D014508:Urea / Q000737:chemistry
D002021:Buffers; D010446:Peptide Fragments; D010743:Phospholipids; D020033:Protein Isoforms; D020710:RGS Proteins; D011993:Recombinant Fusion Proteins; D012492:Salts; C116925:regulator of g-protein signaling 9; D014508:Urea