Characterization of subcellular localization and stability of a splice variant of G alpha i2.
BACKGROUND
Alternative mRNA splicing of alpha(i2), a heterotrimeric G protein alpha subunit, has been shown to produce an additional protein, termed salpha(i2). In the salpha(i2) splice variant, 35 novel amino acids replace the normal C-terminal 24 amino acids of alpha(i2). Whereas alpha(i2) is found predominantly at cellular plasma membranes, salpha(i2) has been localized to intracellular Golgi membranes, and the unique 35 amino acids of salpha(i2) have been suggested to constitute a specific targeting signal.


RESULTS
This paper proposes and examines an alternative hypothesis: disruption of the normal C-terminus of alpha(i2) produces an unstable protein that fails to localize to plasma membranes. salpha(i2) is poorly expressed upon transfection of cultured cells; however, radiolabeling indicated that alpha(i2) and salpha(i2) undergo myristoylation, a co-translational modification, equally well suggesting that protein stability rather than translation is affected. Indeed, pulse-chase analysis indicates that salpha(i2) is more rapidly degraded compared to alpha(i2). Co-expression of betagamma rescues PM localization and increases expression of salpha(i2). In addition, alpha(i2)A327S, a mutant previously shown to be unstable and defective in guanine-nucleotide binding, and alpha(i2)(1-331), in which the C-terminal 24 amino acids of alpha(i2) are deleted, show a similar pattern of subcellular localization as salpha(i2) (i.e., intracellular membranes rather than plasma membranes). Finally, salpha(i2) displays a propensity to localize to potential aggresome-like structures.


CONCLUSIONS
Thus, instead of the novel C-terminus of salpha(i2) functioning as a specific Golgi targeting signal, the results presented here indicate that the disruption of the normal C-terminus of alpha(i2) causes mislocalization and rapid degradation of salpha(i2).
Wedegaertner
Philip B
PB
Department of Microbiology and Immunology Kimmel Cancer Institute Thomas Jefferson University 233 S, 10th St, 839 BLSB Philadelphia, PA 19107, USA. P_Wedegaertner@mail.jci.tju.edu
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D019556:COS Cells / Q000737:chemistry; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D008565:Membrane Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go