Abstract
Rab proteins are membrane-bound prenylated GTP-binding proteins required for the targeted movement of membrane vesicles from one organelle to another. In the current paper we have characterized and purified an enzyme that attaches geranylgeranyl residues to Rab proteins that bear the COOH-terminal sequence Cys-X-Cys (such as Rab3A) and Cys-Cys (such as Rab1A). This enzyme is designated Rab geranylgeranyl transferase (Rab GG transferase). At high salt concentrations, Rab GG transferase from rat brain cytosol separates into two components, designated A and B, both of which are required for activity. We purified Component B to apparent homogeneity and found that it contains two peptides of 60 and 38 kDa. The purified Rab GG transferase did not attach geranylgeranyl to p21H-ras-CVLL, which is prenylated by a GG transferase of the CAAX type that resembles the CAAX farnesyltransferase. Rab GG transferase was strongly inhibited by Zn2+, a cation that is absolutely required by farnesyltransferase. The Rab GG transferase was also inhibited by NaCl concentrations in excess of 100 mM. Together with previous data, the current findings indicate that mammalian cells possess at least three protein prenyltransferases (CAAX farnesyltransferase, CAAX GG transferase, and Rab GG transferase) that are specific for different classes of low molecular weight GTP-binding proteins and other proteins.
Highlights
From the $Departmentof Molecular Genetics ana!the THoward Hughes Medical Institute, University of Teras Southwestern Medical Center, Dallas, Texas 75235
Rab GG transferase from rat brain cytosol separates into two components, designated A and B, both of which are required for activity
Fractions were assayed for Rab GG transferase activity by trichloroacetic acid precipitation following incubation with [3H]GGPP anrdecombinant Rab3A isolated from E. coli
Summary
From the $Departmentof Molecular Genetics ana!the THoward Hughes Medical Institute, University of Teras Southwestern Medical Center, Dallas, Texas 75235. Most of binding proteins required for the targeted movement the known Rab proteins terminate in either of two sequences: of membrane vesicles from one organelle to another. In the current paper we have characterized and purified an enzyme that attaches geranylgeranyl residues to Rab proteins that bear theCOOH-terminalsequence Cys-X-Cys(such as Rab3A) and Cys-Cys (such as RablA). This enzyme is designated Rab geranylgerany transferase (Rab GG transferase). In addition to Rab proteins, other small membrane-bound GTP-binding proteins, such as the ~ 2 1 "pr~oteins, are prenylated, asareother extrinsic membrane proteins, such as nuclear lamins and the y-subunoitf heterotrimeric G proteins. Rab GG transferase was ment of either 20-carbon geranylgeranyl or 15-carbon farnesyl strongly inhibited by Zn2+,a cation that is absolutely groups to cysteine residues in these proteins
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