Isolation and proteomic analysis of rice Golgi membranes: Cis-Golgi membranes labeled with GFP-SYP31

Abstract

The Golgi membranes labeled with cis-Golgi marker GFP-SYP31 were isolated from suspension-cultured cells of rice transformed with 35S::GFP-SYP31 by floating through a discontinuous sucrose density gradient in the presence of 5 mM MgCl2. The specific fluorescence intensity of final membrane preparation increased to approximately 150-fold in comparison with that of the post-nucleus soluble fraction. Specific activity of membrane-bound α-mannosidase (cis-Golgi) markedly increased, but NDPase (medial-/trans-Golgi) and NADPH-cytochrome c reductase endoplasmic reticulum were weakly detected in the membrane fraction. The other organelle marker enzymes, cytochrome c oxidase (mitochondria), alkaline pyrophosphatase (plastid), and catalase (peroxisome) were not detectable. Comparative display of protein spots in GFP-SYP31-labeled membranes, microsomal membranes and soluble fraction on the two dimensional gels showed that some proteins are markedably concentrated in the cis-Golgi membrane fraction. Furthermore, the mass spectrometric analysis of proteins separated by SDS-polyacrylamide gel electrophoresis indicated that the highly purified Golgi membranes contained several membrane traffic-related proteins and ER resident proteins. These findings indicated a close relationship between the ER and the cis-Golgi membranes. Copyright © 2006 The Japanese Society for Plant Cell and Molecular Biology.