A proline-rich region and nearby cysteine residues target XLalphas to the Golgi complex region.
XLalphas is a splice variant of the heterotrimeric G protein, Galpha(s), found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XLalphas for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XLalphas incorporated [(3)H]palmitate, and mutation of cysteines in a cysteine-rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XLalphas without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a Galpha(i2) splice variant, dynamin II, betaIII spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein alpha subunits and possibly for other peripheral membrane proteins as well.
Ugur
O
O
Department of Pharmacology, Medical Faculty, Ankara University, 06100 Ankara, Turkey.
Jones
T L
TL
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002864:Chromogranins; D003545:Cysteine / Q000737:chemistry*; D019205:GTP-Binding Protein alpha Subunits, Gs; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D016716:PC12 Cells; D019308:Palmitic Acid / Q000378:metabolism; D011392:Proline / Q000737:chemistry*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D002864:Chromogranins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019308:Palmitic Acid; D011392:Proline; C406732:GNAS protein, human; C498643:Gnas protein, rat; D019205:GTP-Binding Protein alpha Subunits, Gs; D020962:Heterotrimeric GTP-Binding Proteins; D003545:Cysteine