The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane.
Plasmids containing cDNAs encoding either the wild-type guanine-nucleotide-binding protein G(o)1 alpha or the palmitoylation-negative cysteine-3-to-serine (C3S) mutant of G(o)1 alpha were transfected into Rat 1 cells, and clones stably expressing immunoreactivity corresponding to these polypeptides were isolated. Clones C5B (expressing wild-type G(o)1 alpha) and D3 (expressing the mutant form) were selected for detailed study. Immunoprecipitation of whole cell lysates of each clone labelled with either [3H]palmitate or [3H]myristate demonstrated incorporation of [3H]myristate into both wild-type and the C3S mutant of G(o)1 alpha, but that incorporation of hydroxylamine-sensitive [3H]palmitate was restricted to the wild type. When membrane and cytoplasmic fractions were prepared from cells of either the C5B or D3 clones, although immunodetection of wild-type G(o)1 alpha was observed only in the membrane fraction, the C3S mutant was present in both membrane and cytoplasmic fractions. Furthermore, a significant proportion of the C3S G(o)1 alpha immunoreactivity was also detected in the cytoplasmic fraction if immunoprecipitation of recently synthesized G(o)1 alpha was performed from fractions derived from cells pulse-labelled with [35S]Trans label. Pretreatment of cells of both clones C5B and D3 with pertussis toxin led to complete ADP-ribosylation of the cellular population of G(o)1 alpha in both cell types, irrespective of whether the polypeptide was subsequently found in the membrane or cytoplasmic fraction following cellular disruption. By contrast, separation of membrane and cytoplasmic fractions before pertussis-toxin-catalysed [32P]ADP-ribosylation allowed modification only of the membrane-associated G(o)1 alpha (whether wild-type or the C3S mutant). This labelling was decreased substantially by incubation of the membranes with guanosine 5'-[beta gamma-imido]triphosphate. No cytoplasmic G-protein beta subunit was detected immunologically, and the non-membrane-associated C3S G(o)1 alpha from D3 cells migrated as an apparently monomeric 40 kDa protein on a Superose 12 gel-filtration column. Membrane-associated wild-type and C3S G(o)1 alpha appeared to interact with guanine nucleotides with similar affinity, as no alteration in the dose-response curves for guanine-nucleotide-induced maintenance of a stable 37 kDa tryptic fragment was noted for the two forms of G(o)1 alpha. Chemical depalmitoylation of membranes of clone C5B with neutral 1 M hydroxylamine caused a release of some 25-30% of each of G(o)1 alpha, Gi2 alpha and Gq alpha/G11 alpha from the membranes. Equivalent treatment of D3 cells caused an equivalent release of Gi2 alpha and Gq alpha/G11 alpha, but was unable to cause any appreciable release of the CS3 form of G(o)1 alpha, which was membrane-bound.
Grassie
M A
MA
Department of Biochemistry, University of Glasgow, U.K.
McCallum
J F
JF
Guzzi
F
F
Magee
A I
AI
Milligan
G
G
Parenti
M
M
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003545:Cysteine; D005347:Fibroblasts; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D015151:Immunoblotting; D009154:Mutation; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism*; D010168:Palmitates / Q000378:metabolism*; D037342:Pertussis Toxin; D011233:Precipitin Tests; D051381:Rats; D012694:Serine; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D006898:Hydroxylamines; D009227:Myristic Acids; D010168:Palmitates; D010566:Virulence Factors, Bordetella; D019814:Myristic Acid; D019811:Hydroxylamine; D012694:Serine; D000244:Adenosine Diphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D003545:Cysteine