Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport.

Abstract

N-Ethylmaleimide (NEM) inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. After inactivation of the Golgi membranes by NEM, transport can be rescued by adding back an appropriately prepared cytosol fraction. This complementation assay has allowed us to purify the NEM-sensitive factor, which we term NSF. The NEM-sensitive factor is a tetramer of 76-kDa subunits, and appears to act catalytically, one tetramer leading to the metabolism of numerous transport vesicles.The movement of proteins between membrane-bound compartments is carried out by transport vesicles (1). Elucidation of the molecular mechanisms involved in vesicle budding and fusion will require that the components of the transport machinery be isolated in functional form. Here, we report the purification to essential homogeneity of one such protein component. This protein, the N-ethylmaleimide-sensitive factor (NSF), is needed for biosynthetic transport between Golgi cisternae in cell-free systems (2, 3).Biosynthetic protein transport occurs in two distinct phases. First, a chain of nonselective "bulk-carrier" vesicles move proteins from the endoplasmic reticulum to the Golgi, from cisternae to cisternae across the Golgi stack, and into the trans-Golgi network (4). Then, in the trans-Golgi network, the proteins are separated according to their destinations (5-7). Much of the first part of this pathway has been reconstituted in a cell-free system that measures the transport of the vesicular stomatitis virus-encoded glycoprotein (VSV-G protein) between successive cisternae of the Golgi stack (8-13). Transport requires ATP as well as cytosol (high-speed supernatant), and appears to be mediated by non-clathrincoated vesicles that are the bulk carriers mentioned above (12,14).We have reported (2) that treatment with N-ethylmaleimide (NEM) under mild conditions (1 mM, 15 min, 0°C) selectively inactivated Golgi membranes in the cell-free transport assay. Transport could be restored to NEM-treated Golgi membranes by adding back the ATP extract of untreated Golgi membranes. This restorative factor was itself sensitive to NEM. The activity of NSF is stimulated by long chain fatty acyl-CoA (2, 3). NSF is required for transport at multiple levels of the Golgi stack (3). Here we report the purification of NSF from CHO cytosol prepared in the presence of ATP.MATERIALS AND METHODS Preparation of Golgi Membranes and NSF-Free Cytosol. Donor and acceptor membranes were prepared as described (8,15) The NSF fraction to be tested (up to 20 pl) was added last. After incubation at 370C for 1 hr, the VSV-G protein was immunoprecipitated at 40C for at least 6 hr as described (8). Assays of all fractions were performed in a predetermined linear range (0-0.2 mg/ml for crude ATPstabilized cytosol). Large-Scale Preparation of ATP-Containing Cytosol for NSF Purifications. A washed pellet of CHO cells (1 vol) was resuspended with 4 vol of swelling buffer containing 20 mM Pipes-KOH (pH 7.2), 10 mM MgCl2, 5 mM ATP, 5 mM dithio...

Reconstitution of constitutive secretion using semi-intact cells: regulation by GTP but not calcium.

“…The reaction requires hydrolyzable ATP, exogenous cytosolic components provided by a bovine brain cytosol fraction, and is inhibited by the NEM. It is presently unknown how many factors are inhibited by NEM, and whether the NEM-sensitive factor, which has been shown to participate in the fusion reactions of ER to Golgi , intra-Golgi (Block et al, 1988), and endosomal transport , is involved in this step of transport. Future experiments using our system should resolve this question.…”

Secretion machinery at the cell plasma membrane

“…A primary understanding of membrane fusion in cells was made possible following discovery of a N-ethylmaleimide-sensitive factor (NSF) [35] and SNARE proteins [14][15][16], and their involvement in membrane fusion [36,37]. Target membrane proteins, SNAP-25 and syntaxin (t-SNARE) and secretory vesicle-associated membrane protein (VAMP or v-SNARE), are part of the conserved protein complex involved in fusion of opposing bilayers [37].…”

N-Ethylmaleimide-sensitive Fusion Protein Contains High and Low Affinity ATP-binding Sites That Are Functionally Distinct

“…The N-ethylmaleimide-sensitive factor (NSF) 1 was originally purified based on its ability to restore intercisternal transport activity to Golgi membranes that had been previously treated with the alkylating agent N-ethylmaleimide (NEM) (1,2). The cDNA encoding this protein was subsequently cloned (3) and shown to have significant homology to a previously identified yeast gene, Sec18 (4).…”