Proc. Nati. Acad. Sci. USA Vol. 85, pp. 7852-7856, November 1988
MARC R. BLOCK, BENJAMIN S. GLICK, CELESTE A. WILCOX, FELIX T. WIELAND, AND JAMES E. ROTHMAN*
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 (VSVG 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) from VSV-infected 15B CHO and wild-type uninfected CHO cells, respectively. A mixture of equal volumes of acceptor and donor Golgi membranes (in 1 M sucrose/10 mM Tris·HCI, pH 7.4) was incubated at 0°C for 15 min with 1 mM NEM (final concentration) added from a fresh stock solution (50 mM). Then dithiothreitol was added to 2 mM from a 0.1 M stock. This mixture of NEM-treated donor and acceptor membranes was refrozen in liquid N2 and stored in aliquots at - 80°C for use in NSF assays. 15B CHO cells were grown and homogenized (15). Cytosol (8 mg/ml of protein) was prepared and desalted on a Bio-Gel P6-DG column essentially as described (8, 15), incubated for 20 min at 37°C in the absence of ATP to inactivate any NSF, frozen in liquid nitrogen, and stored at - 80°C.
Assay of NSF Activity. The assay used is a variation on the standard transport assay (8). Incubation mixtures (50 μl) contained NEM-treated donor (5 μl) and acceptor (5 μl) membranes, 5 μl of NSF-free 15B CHO cytosol, 10 AM palmityl-CoA, 50 μM ATP, 2 mM creatine phosphate, creatine kinase (7.3 international units/ml), 250 μM UTP, and 0.4 μM UDP-[3H]GlcNAc (0.5 uCi; 1 Ci = 37 GBq) in an assay buffer containing 25 mM Hepes-KOH (pH 7.0), 15 mM KCI, 2.5 mM Mg(OAc)2, and 0.2 M sucrose (derived from the Golgi fractions). The NSF fraction to be tested (up to 20 μl) was added last. After incubation at 37°C for 1 hr, the VSV-G protein was immunoprecipitated at 4°C 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 ATP-stabilized 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 dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM ο-phenanthroline, leupeptin (10 μ/ml), and 1 μM pepstatin. The cells were allowed to swell 20 min on ice and then were disrupted in a Waring Blendor at high speed for 30 sec. Then, KCI was added slowly to the homogenate while stirring to achieve a final concentration of 0.1 M from a 2.5 M stock solution. After low-speed centrifugation at 800 x g for 15 min, the postnuclear supernatant was frozen in 45-ml aliquots in liquid nitrogen and stored at - 80°C. When cytosol was required, postnuclear supernatant (rapidly thawed at 37°C) was spun at 45,000 rpm in a 45 Ti rotor for 90 min. This supernatant was the cytosol used for purification. Purification of NSF. Fresh dithiothreitol was added to all buffers immediately before use. The purification described here is a standard one designed for 450 ml of cytosol and employs tandem DE-52 (Whatman) and S Sepharose Fast Flow (Pharmacia) columns. The purification described in the text and Table 1 and Figs. 2 and 3 employed 120 ml of cytosol (600 mg of protein) and is meant to illustrate each step separately. The various steps can be scaled up or down in a straightforward proportional manner as needed.