Expression and localization of rab escort protein isoforms in parotid acinar cells from rat.
Rab proteins are geranylgeranylated on their carboxyl terminal cysteine motifs by geranylgeranyltransferase II (GGTase). Rab escort protein (REP) is required to present Rab proteins to GGTase. REP may remain bound to newly isoprenylated Rab proteins and present them to their target membrane. Other studies have shown that Rab proteins cycle between the membrane and cytosolic compartments and that cytosolic Rab proteins are complexed with rab-GDI. In the present study, we examined the expression and localization of REP isoforms in parotid acinar cells. Although both REP isoforms, REP-1 and REP-2, were detected in parotid cytosol, REP-2 was the predominant isoform. Subcellular fractionation revealed that approximately 42% of cellular REP-2 is membrane-associated. REP-2 was partially removed from parotid membranes with 1 M NaCl or Na(2)CO(3), indicating that REP-2 is a peripheral membrane protein. Membrane-associated REP-2 did not colocalize with Rab3D on secretory granule membranes. However, density gradient centrifugation revealed that membrane-associated REP-2 and Rab3D colocalize on low- and high-density membrane fractions in parotid acinar cells. Isoproterenol, an agent which induces amylase release from parotid glands, caused a shift in both REP-2 and Rab3D to less dense membrane fractions. When acinar cell cytosol was fractionated by gel filtration chromatography, Rab3D eluted exclusively with REP, not rab-GDI. In contrast, Rab1B and Rab5 eluted with both REP and Rab-GDI. Colocalization of Rab3D and REP-2 on acinar cell membranes suggests that REP-2 plays a role in delivering Rab3D to parotid membranes and may regulate guanine nucleotide binding to membrane-associated Rab3D. In addition, unlike other Rab proteins, cytosolic Rab3D appears to associate exclusively with REP, not rab-GDI in parotid acinar cells.
Chan
D
D
Division of Digestive Diseases, Department of Medicine, State University of New York-Health Science Center at Brooklyn, Brooklyn, New York, USA.
Lin
J
J
Raffaniello
R D
RD
D048868:Adaptor Proteins, Signal Transducing; D019883:Alkyl and Aryl Transferases; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D003594:Cytoplasmic Granules; D005089:Exocytosis; D008297:Male; D010306:Parotid Gland / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D017368:Protein Prenylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D020691:rab GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C513166:Chm protein, rat; D020033:Protein Isoforms; D019883:Alkyl and Aryl Transferases; D020691:rab GTP-Binding Proteins