C H A P T E R 29 Microsome-B, ased Assay for Analysis of Endoplasmic Reticulum to Golgi Transport in Mammalian Cells Helen Plutner, Cemal Gurkan, Xiaodong Wang, Paul LaPointe, and William E. Balch The trafficking of proteins along the first stage of the secretory pathway is mediated by small vesicles that bud from the endoplasmic reticulum (ER) and subse- quently fuse with the cis-Golgi compartment. This article describes a biochemical assay using mammalian microsomes that can be used to measure these events independently. The microsomes are prepared from cells infected at the restrictive temperature (39.5~ with the ts045 strain of vesicular stomatitis virus (VSV) (Lafay, 1974). As a reporter molecule the assay utilizes ts045 VSV-glycoprotein (VSV-G), which is retained in the ER during infection due to a thermoreversible folding defect; incubation in vitro at the permissive temperature (32~ results in the synchronous folding and transport of VSV-G to the Golgi complex. To follow vesicle forma- tion, a differential centrifugation procedure is employed to separate the more rapidly sedimenting ER and Golgi membranes from the slowly sedimenting vesicles. Con- sumption is analyzed using a two-stage assay in which vesi- cles isolated by differential centrifugation during stage 1 are subsequently added to stage 2 (fusion) reactions containing acceptor Golgi membranes. Transport to the Golgi is measured by following the oligosaccharide processing of VSV-G from the high mannose ER form, which is sensitive to endoglycosidase H (endo H), to the cis/medial-Golgi form, which is endo H resistant (Schwaninger et al., 1992). The biochemical characteris- tics of the overall ER to Golgi transport reaction and the vesicle formation and consumption assays are described elsewhere (Aridor et al., 1995,1996b, 1998,1999a,b, 2000, 2001; Rowe et al., 1996a). II. MATERIALS AND INSTRUMENTATION Culture medium (0~-MEM; Cat. No. 11900-099) is from Life Technologies. The medium is supplemented with penicillin/streptomycin from a 100x stock solu- tion (Cat. No P0781; Sigma). Fetal bovine serum (FBS; Cat. No FB-01) is from Omega Scientific. D-Sorbitol (Cat. No. S-1876), leupeptin (Cat. No L-2884), chymo- statin (Cat. No C-7268), pepstatin (Cat. No P-4265), phenylmethylsulfonyl fluoride (PMSF; Cat. No P- 7626), actinomycin D (Cat. No A-1410), uridine 5'- diphospho-N-acetylglucosamine (UDP-GlcNAc; Cat. No. U-4375), and dimethyl sulfoxide (DMSO; Cat. No. D-2650), are from Sigma. The nitrocellulose mem- brane (Cat. No. 68260) is from Schleicher & Schuell. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Cat. No. 31460) is from Pierce. Chemilumines- cence reagent (Cat. No. NEL-101) and autoradiogra- phy film ("Reflection") are from NEN. Polyallomer microfuge tubes (Cat. No. 357448) are supplied by Beckman Instruments Inc. A polyclonal antibody to VSV-G is generated in rabbits immunized with the C-terminal 16 amino acids of VSV-G (Indiana serotype) coupled to KLH (Plutner et al., 1991). Centrifugation at 20,000 or 100,000g is performed using an Optima TL ultracentrifuge (Beckman) equipped with a TLA 100.3 rotor. A laser-scanning densitometer (personal densit- ometer; Molecular Dynamics) is used to quantitate data. Cell Biology 209 Copyright 2006, Elsevier Science (USA). All rights reserved.