Abstract
Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library. They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily. The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized. Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities. H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart. The other proteins share approximately 40% homology with YPT1p and SEC4p. The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding. Human rab proteins were produced in Escherichia coli. Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents. All six proteins bind GTP and exhibit GTPase activities. A possible involvement of the rab proteins in secretion is discussed.
Highlights
Seven cDNA clones corresponding to the rabl, rab2, rab3A, rab3B, rab4, rab5, and rub6 genes were isolated from a human pheochromocytomacDNA library
Ampicillin, with an additional 12.5 pg/ml chloramphenicolin the case of BL21 pLysS. They were diluted to 1:50 in LB medium containing 50 pg/ml Ampicillin andincubated for 1 h a t 37 "C followed by induction with 200 p~ isopropyl-(3-D-thiogalactosidfeor 3 h. 1 ml of the bacterial suspension was centrifuged and the pellet sequenced by the dideoxy chainterminationmethod.The nucleotide and deduced amino acid sequences of the human H-rabl, 2,3A, and4 cDNAs are shown in Fig. 1.The initiator ATGcodons were determined by alignment of the human resuspended in 200 p1 of SDS sample buffer; the extract was boiled sequences with the published rat sequences as well as from for5 min and 20 pl subjected to SDS-PAGE electrophoresis(29). computer analysis.Nucleotidesequencesreveallongopen
In this report,we describe the identification of their human homologs (H-rabl, H-rab2, H-rab3A, and H-rab4) and of three additionalrub cDNAs (H-rab3B, Hrab5, and H-rab6)
Summary
Containing the complete coding sequence of each rab cDNA were cloned in appropriate sites of M13 vectors to create a HindIII (2.5 kbp for rabl, 1.20 kbp for rab2, 1.35 kbp for rab3A, and Bacteria were grown overnight in LB medium containing 100 pg/ml 1.4 kbpfor rub were selected and the inserts completely. Ampicillin, with an additional 12.5 pg/ml chloramphenicolin the case of BL21 pLysS They were diluted to 1:50 in LB medium containing 50 pg/ml Ampicillin andincubated for 1 h a t 37 "C followed by induction with 200 p~ isopropyl-(3-D-thiogalactosidfeor 3 h. Purification of Rab Proteins-250 ml of an overnight culture of HBlOl orBL21ply& containingtheappropriate plasmidin LB medium (with 50 pg of ampicillin/ml) were inoculated in 5 liters of reading frames coding for 205(H-rabl), 212(H-rab2), 220(Hrab3A), and 213(H-rab4) amino acid proteins of respective predicted molecular weight663 632912, and 23,875.
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