title: Targeting of chimeric G alpha i proteins to specific membrane domains.
abstract: Heterotrimeric guanine nucleotide-regulatory (G) proteins are associated with a variety of intracellular membranes and specific plasma membrane domains. In polarized epithelial LLC-PK1 cells we have shown previously that endogenous G alpha i-2 is localized on the basolateral plasma membrane, whereas G alpha i-3 is localized on Golgi membranes. The targeting of these highly homologous G alpha i proteins to distinct membrane domains was studied by the transfection and expression of chimeric G alpha i proteins in LLC-PK1 cells. Chimeric cDNAs were constructed from the cDNAs for G alpha i-3 and G alpha i-2 and introduced into a pMXX eukaryotic expression vector containing a mouse metallothionein-I promoter. Stably transfected cell lines were produced that expressed either G alpha i-2/3 or G alpha i-3/2 chimeric proteins. Chimeric and endogenous G alpha i proteins were detected in cells using specific carboxy-terminal peptide antibodies. Immunofluorescence staining was used to localize endogenous and chimeric G alpha i proteins in LLC-PK1 cells. The staining of chimeric proteins was detected as an increased intensity of staining on membranes containing endogenous G alpha i proteins. Using confocal microscopy and image analysis we localized G alpha i-2 to a specific sub-domain of the lateral membrane of polarized cells, the chimeric G alpha i-3/2 protein was then shown to colocalize with endogenous G alpha i-2 in the same lateral plasma membrane domain. The chimeric G alpha i-2/3 protein colocalized with endogenous G alpha i-3 on Golgi membranes in LLC-PK1 cells. These results show that chimeric G alpha i proteins were targeted to the same membrane domains as endogenous G alpha i proteins and the specificity of their membrane targeting was conferred by the carboxy-terminal end of the proteins. These data provide the first evidence for specific targeting information contained in the carboxy termini of G alpha i proteins, which appears to be independent of amino-terminal membrane attachment sites in these proteins.
authors.affiliation: Renal Unit, Massachusetts General Hospital, Boston 02129.
authors.forename: J B
authors.initials: JB
authors.lastname: de Almeida
authors.forename: E J
authors.initials: EJ
authors.lastname: Holtzman
authors.forename: P
authors.initials: P
authors.lastname: Peters
authors.forename: L
authors.initials: L
authors.lastname: Ercolani
authors.forename: D A
authors.initials: DA
authors.lastname: Ausiello
authors.forename: J L
authors.initials: JL
authors.lastname: Stow
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D004247:DNA; D019204:GTP-Binding Proteins