Two‐dimensional mapping of the endogenous proteins of the rat hepatocyte Golgi complex cleared of proteins in transit

Abstract

The discovery of additional endogenous Golgi proteins will lead to significant new insights into Golgi function. To this end, stacked Golgi fractions (SGFs) were isolated from rat liver before (CTL SGF) and after molecules in transit through the Golgi were cleared by pre-treatment with cycloheximide (CHX SGF). Electron microscopic (EM) morphometric and biochemical analyses showed that the in vivo stacked morphology is retained, that > 90% of the elements can be positively identified as Golgi stacks and cisternae, and that transmembrane protein markers of the Golgi complex are enriched 300- to 800-fold over starting postnuclear supernatant (PNS). High-resolution two-dimensional (2-D) gel mapping has been carried out on the CTL PNS, CTL SII (an intermediate fraction), CTL SGF, CHX SGF, CHX SGF - high pH supernatant, and CHX SGF - high pH pellet. This analysis, coupled with immunoblotting and alignment of the 2-D gels with master gels, has allowed the identification of a number of known proteins and the preliminary characterization of the most abundant 173 Golgi-specific proteins. These 173 proteins have been placed into three categories: cargo, cytosolic Golgi-associated, and resident Golgi proteins. These categories are tentative and will be modified as more data are acquired from immunoblotting and protein sequencing.

Analysis of the Arabidopsis nuclear proteome and its response to cold stress

“…Fractionation of a proteome into subproteomes is based on the macromolecular architecture of the cell, including subcellular compartments, organelles, macromolecular structures, and multiprotein complexes (Jung et al, 2000a). To date, 2D electrophoresis reference maps have been constructed for speci®c organelles, including mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi apparatus, and nucleus (Bell et al, 2001;Chataway et al, 1998;Fialka et al, 1999;Jung et al, 2000b;Rabilloud et al, 1998;Taylor et al, 1997).…”

Proteomic Analysis of Two Functional States of the Golgi Complex in Mammary Epithelial Cells

“…Given the many complex functions of the organelle, some of which remain unknown, the Golgi complex has been an area of intense research in the last 20 years. It is estimated that at least 1000 proteins make up the protein complement of the Golgi complex, and, to date, less than 200 of these are known (10). Clearly, efficient screens revealing novel Golgi proteins and protein interactions would enhance our present understanding of Golgi function.…”

The Current State of the Golgi Proteomes

“…The study used cyclohexamide in an effort to clear transitory proteins from the secretory pathway and reduce non-specific Golgi proteins. While only a handful of proteins were identified by cross comparing reference maps and immunoblotting the study demonstrated the validity of a proteomic approach to analyze the Golgi and could discern resident proteins from cargo and cytosolic proteins (Taylor et al, 1997b). Significantly, the study employed a recently developed sequential sucrose gradient enrichment method (Taylor et al, 1997a) and enabled the reliable visualization of Golgi proteins by 2-DE with reduced contaminants.…”

“…Nearly all of the early proteomic studies on enriched Golgi fractions are from easily accessible samples such as rat livers, likely reflecting the need for the development of purification techniques. The earliest 'proteomic' analyses of the Golgi employed two-dimensional gel electrophoresis (2-DE) to array enriched stacked Golgi fractions from rat livers (Taylor et al, 1997b). The study used cyclohexamide in an effort to clear transitory proteins from the secretory pathway and reduce non-specific Golgi proteins.…”