Abstract
Trafficking and sorting of lipids during transport from the endoplasmic reticulum to the Golgi apparatus was studied using a cell-free system from rat liver. Transitional elements of the endoplasmic reticulum were prepared from liver slices prelabeled with [14C]- or [3H]acetate as the donor fraction. Non-radioactive Golgi apparatus were immobilized on nitrocellulose as the acceptor. When reconstituted, the radiolabeled donor retained a capacity to transfer labeled lipids to the non-radioactive Golgi apparatus acceptor. Transfer exhibited two kinetically different components. One was stimulated by ATP, facilitated by cytosol and inhibited by guanosine 5'-O-(thiotriphosphate) and N-ethylmaleimide. In parallel with protein transport, the ATP-dependent lipid transfer occurred with a temperature transition at about 20 degrees C. The other was not stimulated by ATP, did not require cytosol, was acceptor unspecific, was unaffected by inhibitors and, while temperature dependent, did not exhibit a sharp temperature transition. The ATP-independent transfer was non-vesicular. In contrast, the ATP-dependent transfer was vesicular. Transition vesicles isolated by preparative free-flow electrophoresis, when used as the donor fraction, transferred lipids to Golgi apparatus acceptor with a 5-6-fold greater efficiency than that exhibited by the unfractionated transitional endoplasmic reticulum. Formation of transition vesicles was ATP-dependent. Transferred lipids were chiefly phosphatidylcholine and cholesterol. Membrane triglycerides, major constituents of the transitional endoplasmic reticulum membranes, were both depleted in the transition vesicle-enriched fractions and not transferred to Golgi apparatus suggestive of lipid sorting prior to or during transition vesicle formation. The characteristics of the ATP plus cytosol-dependent transfer were similar to those for protein transfer mediated by transition vesicles. Thus, the 50-70-nm vesicles derived from transitional endoplasmic reticulum appear to function in the trafficking of both newly synthesized proteins and lipids from the endoplasmic reticulum to the Golgi apparatus.
Highlights
Trafficking and sorting of lipids during transport membrane constituents from the endoplasmicreticulum to from theendoplasmic reticulumto theGolgi apparatus the cell surface has come from studies of the biosynthesis, was studied using a cell-free system from rat liver. processing, sorting, and transport of membrane or secretory
In order to determine which lipids were transported by the 17) that bleb from part-rough, part-smoothtransition elevesicular pathway, the distributions of labeled lipids of tran- ments of the endoplasmic reticulum [17]. They fuse at sitional endoplasmic reticulum membranes and of the tran- the cis face of the Golgi apparatus either to form new Golgi sition vesicles isolated by free-flowelectrophoresis (Table IV) apparatus cisternaeor to merge with existing Golgi apparatus were compared
The system we describe from liver utilizes purified transition endoplasmic reticulum and Golgi apparatus fractions
Summary
0.33 k 0.04 0.35 f 0.04 ratus and nogtiven by either endoplasmic reticulum or plasma membraneasacceptor(Table 111). The ATP-dependent transport of lipids from the transitional endoplasmic reticulumtothe Golgi apparatus was inhibited significantly by 40 PM GTPrS (Table 11). When preincubated lipids between labeled transitional endoplasmic reticulum with membranes and cytosol, 1 mM NEM to an extenctomparable to that obtained inhibited transfer with GTPrS In order to determine which lipids were transported by the 17) that bleb from part-rough, part-smoothtransition elevesicular pathway, the distributions of labeled lipids of tran- ments of the endoplasmic reticulum [17] They fuse at sitional endoplasmic reticulum membranes and of the tran- the cis face of the Golgi apparatus either to form new Golgi sition vesicles isolated by free-flowelectrophoresis (Table IV) apparatus cisternaeor to merge with existing Golgi apparatus were compared. Except for sphingomyelin [27], the major membrane phospholipids appear to be synthesized primarily at the endo-
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