Different properties of the native and reconstituted heterotrimeric G protein transducin.
Visual signal transduction serves as one of the best understood G protein-coupled receptor signaling systems. Signaling is initiated when a photon strikes rhodopsin (Rho) causing a conformational change leading to productive interaction of this G protein-coupled receptor with the heterotrimeric G protein, transducin (Gt). Here we describe a new method for Gt purification from native bovine rod photoreceptor membranes without subunit dissociation caused by exposure to photoactivated rhodopsin (Rho*). Native electrophoresis followed by immunoblotting revealed that Gt purified by this method formed more stable heterotrimers and interacted more efficiently with membranes containing Rho* or its target, phosphodiesterase 6, than did Gt purified by a traditional method involving subunit dissociation and reconstitution in solution without membranes. Because these differences could result from selective extraction, we characterized the type and amount of posttranslational modifications on both purified native and reconstituted Gt preparations. Similar N-terminal acylation of the Gtalpha subunit was observed for both proteins as was farnesylation and methylation of the terminal Gtgamma subunit Cys residue. However, hydrogen/deuterium exchange experiments revealed less incorporation of deuterium into the Gtalpha and Gtbeta subunits of native Gt as compared to reconstituted Gt. These findings may indicate differences in conformation and heterotrimer complex formation between the two preparations or altered stability of the reconstituted Gt that assembles differently than the native protein. Therefore, Gt extracted and purified without subunit dissociation appears to be more appropriate for future studies.
Goc
Anna
A
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4965, USA.
Angel
Thomas E
TE
Jastrzebska
Beata
B
Wang
Benlian
B
Wintrode
Patrick L
PL
Palczewski
Krzysztof
K
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D003623:Dark Adaptation; D041961:Deuterium Exchange Measurement; D008958:Models, Molecular; D011499:Protein Processing, Post-Translational; D012374:Rod Cell Outer Segment / Q000737:chemistry / Q000378:metabolism; D015293:Transducin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D015293:Transducin