Characterization of interactions between transducin alpha/beta gamma-subunits and lipid membranes.
The gamma-subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are isoprenylated and alpha-carboxyl methylated at their COOH-terminal cysteine residues. These modifications are necessary for membrane attachment of the beta gamma complex, but a requirement of an additional factor has been proposed for the stable binding. We explored a possible contribution of the blocked amino terminus of beta-subunits of bovine photoreceptor G-protein, transducin (T alpha/T beta gamma = Gt alpha/beta 1 gamma 1), and of three beta gamma complexes (beta 1 gamma 2, beta 1 gamma 3, and beta 1 gamma 7) purified from bovine brains. Structural analyses revealed that every beta 1-subunit has an N-acetylated serine, which is unlikely to contribute to the membrane association. Since neither protease nor heat treatment of photoreceptor membranes affected the membrane binding of T beta gamma, it seems unlikely that rhodopsin (or other membrane proteins) serves as an anchor protein for accepting T beta gamma. In fact, T beta gamma bound to phospholipid large unilamellar vesicles (LUVs), of which the polar head groups strongly influenced the binding: T beta gamma alone showed 2-fold higher binding for negatively charged phosphatidylserine-LUVs than for neutral phosphatidylcholine (PC)-LUVs, while the affinity of T alpha/T beta gamma complex for the phosphatidylserine-LUVs was lower than that for the PC-LUVs. These results indicate that 1) an ionic interaction between T beta gamma and membrane surface plays an important role in the stable membrane association, and 2) the domain(s) of T beta gamma responsible for the association would be different between trimeric and dissociated states. We also found that synthetic peptides corresponding to the COOH-terminal region of T gamma inhibited T alpha-T beta gamma interaction only when the peptides were isoprenylated. This suggests that the isoprenyl moiety is located at the contact site between the subunits, not at the membrane-binding domain, when T beta gamma is complexed with T alpha.
Matsuda
T
T
Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Japan.
Takao
T
T
Shimonishi
Y
Y
Murata
M
M
Asano
T
T
Yoshizawa
T
T
Fukada
Y
Y
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D010450:Endopeptidases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010743:Phospholipids / Q000378:metabolism*; D011485:Protein Binding; D017368:Protein Prenylation; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013329:Structure-Activity Relationship; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D008081:Liposomes; D046911:Macromolecular Substances; D009842:Oligopeptides; D010446:Peptide Fragments; D010743:Phospholipids; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D010450:Endopeptidases; D015293:Transducin