Degradation of the non-palmitoylated invertebrate visual guanine-nucleotide binding protein, iGq alpha(C3,4A), by the ubiquitin-proteasomal pathway is regulated by its activation and translocation to the cytoplasm.
Light-dependent translocation of invertebrate visual guanine-nucleotide binding protein, iGq alpha, from rhabdomeric membranes to the cytoplasm is one of many mechanisms that contribute to light adaptation in the invertebrate eye. We have previously cloned iGq alpha from a Loligo pealei photoreceptor cDNA library and shown that when expressed in HEK 293T cells it is palmitoylated. In this study we compared the activation, cytoplasmic translocation, and turnover of iGq alpha with that of a non-palmitoylated mutant, iGq alpha(C3,4A). In the HEK 293T cells, muscarinic M1 receptors coupled equally well to iGq alpha and iGq alpha(C3,4A) to activate phospholipase C. Activation of iGq alpha(C3,4A), but not iGq alpha, induced translocation of the alpha subunit from the membrane to cytosol with rapid degradation of the soluble protein resulting in a decreased half-life for iGq alpha(C3,4A) of 10 hours compared to 20 hours for iGq alpha. Degradation of iGq alpha(C3,4A) was inhibited by proteasomal inhibitors but not by inhibitors of lysosomal proteases or calpain. The presence of the proteasomal inhibitor led to the accumulation of polyubiquitinated species of either iGq alpha or iGq alpha(C3,4A). Our results suggest that palmitoylation of iGq alpha is required to maintain membrane association of the protein in its active conformation, and whereas membrane-bound and soluble iGq alpha can be polyubiquitinated, membrane association protects the protein from rapid degradation by the proteasomal pathway.
Go
Lynle
L
Department of Pharmacology, University of Toronto, Ontario, Canada.
Mitchell
Jane
J
D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002461:Cell Line, Transformed; D018679:Cholinergic Agonists / Q000494:pharmacology; D003001:Cloning, Molecular / Q000379:methods; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D049834:Loligo / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D025801:Ubiquitin / Q000378:metabolism*; D014785:Vision, Ocular / Q000187:drug effects / Q000502:physiology*
D018679:Cholinergic Agonists; D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D025801:Ubiquitin; D002217:Carbachol; D046988:Proteasome Endopeptidase Complex; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde