Cell-free analysis of Golgi apparatus membrane traffic in rat liver

Abstract

Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway.

ATP‐Dependent and drug‐inhibited vesicle enlargement reconstituted using synthetic lipids and recombinant proteins

“…The basis for the present study resides in a series of investigations that pointed to enlargement of plant and animal cells as an active process involving physical membrane displacement [20,21] and a development of cell-free systems to study mechanisms of vesicle budding [24]. For example, budding of vesicles from Golgi apparatus exhibited an absolute requirement for ATP.…”

“…6 first published in 1994 [20]. An extensive evaluation of a series of different donor and acceptor combinations involving reduced pyridine nucleotide eventually yielded a NADH oxidase (NOX) activity that was subsequently shown to be both cell surface (plasma membrane) [23] and Golgi apparatus [24] localized and exquisitely tied to the enlargement phase of cell growth [23].…”

Free‐Flow Electrophoretic Analysis of Endosome Subpopulations of Rat Hepatocytes

“…In any case, an organelle‐enriched fraction is subjected to separation by FFE. Although trypsination is omitted, the starting material is, for example, treated with amylase (Morré, ) or reacted with antibodies (Volkl et al, ). As marker enzymes β‐ N ‐acetylglucosaminidase (lysosomes), urate oxidase (peroxisomen), thiamine pyrophosphatase and UDP‐galactose galactosyltransferase (trans‐Golgi) can be used to follow the organelle purification and enrichment.…”

Two-dimensional electrophoresis reveals differential protein expression in high- and low-secreting variants of the rat basophilic leukaemia cell line

“…A spot identified as transitional endoplasmic reticulum ATPase (TER-ATPase) is evident in gels of the lowsecreting, but absent from the high-secreting variants. As reviewed by Morre [39], this protein has been proposed to control ATP-dependent vesicle formation. The function of this protein in the low-secreting variant is at present uncertain.…”