Identification of a novel domain of Ras and Rap1 that directs their differential subcellular localizations.
The small GTPase Ha-Ras and Rap1A exhibit high mutual sequence homology and share various target proteins. However, they exert distinct biological functions and exhibit differential subcellular localizations; Rap1A is predominantly localized in the perinuclear region including the Golgi apparatus and endosomes, whereas Ha-Ras is predominantly localized in the plasma membrane. Here, we have identified a small region in Rap1A that is crucial for its perinuclear localization. Analysis of a series of Ha-Ras-Rap1A chimeras shows that Ha-Ras carrying a replacement of amino acids 46-101 with that of Rap1 exhibits the perinuclear localization. Subsequent mutational studies indicate that Rap1A-type substitutions within five amino acids at positions 85-89 of Ha-Ras, such as NNTKS85-89TAQST, NN85-86TA, and TKS87-89QST, are sufficient to induce the perinuclear localization of Ha-Ras. In contrast, substitutions of residues surrounding this region, such as FAI82-84YSI and FEDI90-93FNDL, have no effect on the plasma membrane localization of Ha-Ras. A chimeric construct consisting of amino acids 1-134 of Rap1A and 134-189 of Ha-Ras, which harbors both the palmitoylation and farnesylation sites of Ha-Ras, exhibits the perinuclear localization like Rap1A. Introduction of a Ha-Ras-type substitution into amino acids 85-89 (TAQST85-89NNTKS) of this chimeric construct causes alteration of its predominant subcellular localization site from the perinuclear region to the plasma membrane. These results indicate that a previously uncharacterized domain spanning amino acids 85-89 of Rap1A plays a pivotal role in its perinuclear localization. Moreover, this domain acts dominantly over COOH-terminal lipid modification of Ha-Ras, which has been considered to be essential and sufficient for the plasma membrane localization.
Nomura
Kazuhiro
K
Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Kanemura
Hoshimi
H
Satoh
Takaya
T
Kataoka
Tohru
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D019308:Palmitic Acid / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017368:Protein Prenylation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry*; D018631:ras Proteins / Q000737:chemistry*
D000596:Amino Acids; D018076:DNA, Complementary; D008055:Lipids; D011994:Recombinant Proteins; D019308:Palmitic Acid; D006160:Guanosine Triphosphate; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins