Detection of GTP-binding proteins in purified derivatives of rough endoplasmic reticulum

Abstract

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [α-32P]GTP [Bhullar and Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 μM-MgCl2. Binding of [α-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 μM-GDP. Either guanosine 5'-[γ-thio]triphosphate or guanosine 5'-[βγ-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 μM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 μg of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [α-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [α-32P]GTP-binding proteins to have similar acid isoelectric points. [α-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [α-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.