Influence of membrane components in the binding of proteins to membrane surfaces.
We have quantified the enhancement of membrane binding of activated and deactivated Galpha(s) and Galpha(q) subunits, Gbetagamma subunits, and phospholipase Cbeta(2) by lipid rafts and by the presence of membrane-associated protein partners. Membrane binding studies show that lipid rafts do not affect the intrinsic membrane affinity of Galpha(q)(GDP) and Galpha(s)(GDP), supporting the idea that these proteins partition evenly between the domains. Visualization of lipid rafts on monolayers by use of a probe that does not enter raft domains shows that neither activated nor deactivated Galpha(q)(GDP) subunits distribute evenly between the raft and nonraft domains, contrary to previous suggestions. Membrane binding of deactivated Galpha(q) and Galpha(s)(GDP) became weaker when Gbetagamma subunits were present, in contrast with the behavior predicted by thermodynamics. However, activated Galpha subunits and phospholipase Cbeta(2) were recruited to membrane surfaces by protein partners by predicted amounts. Our studies suggest that the anomalous behavior seen for deactivated Galpha subunits in the presence of Gbetagamma subunits may be due to conformational changes in the N-terminus and/or occlusion of a portion of its membrane interaction region by Gbetagamma. Even though membrane recruitment was clearly observed for one protein partner, the presence of a second partner of lower affinity did not further promote membrane binding. For these proteins, the formation of larger protein complexes with very high membrane affinities is unlikely.
Philip
Finly
F
Department of Physiology & Biophysics, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
Scarlata
Suzanne
S
D003374:Coumarins / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D054799:Phospholipase C beta; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology; D013816:Thermodynamics; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism*
D003374:Coumarins; D005456:Fluorescent Dyes; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D007527:Isoenzymes; D006153:Guanosine Diphosphate; C030123:coumarin; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D043802:GTP-Binding Protein alpha Subunits, Gq-G11