We report a cell-free system that measures transport-coupled maturation of carboxypeptidase Y (CPY). the principal CPY processing enzyme proteinase A) and acceptor membranes from a yeast strain indicating intercompartmental transfer. Cytosol made from a yeast strain deleted for the gene was less efficient at driving transport. Moreover antibodies against Vps33p (a Sec1 homologue) and Vam3p (a Q-SNARE) inhibited transport >90%. Cytosolic extracts from yeast cells overexpressing Vps33p restored transport to antibody-inhibited assays. This cell-free system has allowed the demonstration of reconstituted intercompartmental transport coupled to the function of a gene product. (Swanson et al. 1998) (Brenner 1974) and (Novick and Schekman 1979; Deshaies and Schekman 1987) have not only uncovered hundreds of genes but also helped reveal the ubiquitous nature of secretion and endocytosis among eukaryotic organisms. These genetic efforts were pioneered in yeast and include >20 secretion (contains not only a lysosome-like vacuole but also a PLC-like prevacuolar compartment (PVC) (Raymond et al. 1990; Davis et al. 1993; Vida et al. 1993). The PVC is central to the function of nearly all genes. The mutants either cause defects in anterograde or retrograde sorting/transport between the late Golgi complex and the PVC or in transport between the PVC and the vacuole (Bryant and Stevens 1998). Subsequent studies have focused on Hederasaponin B uncovering the function of genes and are beginning to reveal aspects on the biochemistry Hederasaponin B of transport to and from the PVC because several of the gene products have biochemical activity in vitro. For example the gene product is a dynamin-like protein that can bind and hydrolyze (Vater et al. Hederasaponin B 1992). Likewise the gene encodes a protein that binds and hydrolyzes GTP but RCAN1 with similarity to the small G protein Rab5 (Horazdovsky et al. 1994). The gene product is an ATPase (Babst et al. 1997) encodes a phosphatidylinositol-3 kinase (Stack Hederasaponin B and Emr 1994) and Vps15p is a serine/threonine protein kinase (Herman and Emr 1990; Stack et al. 1993). However coupling the catalytic activity of a gene product to intercompartmental protein transport in a reconstituted assay remains elusive. A permeabilized yeast spheroplast system (Vida Hederasaponin B et al. 1990) has not allowed analysis of gene product function because the membranes are not depleted for any protein that has been examined. In this report we describe an intercompartmental protein transport assay using partially Hederasaponin B purified organelles. This cell-free system measures proteolytic maturation of soluble vacuolar proenzymes such as carboxypeptidase Y and proteinase A after transfer from the PVC to the vacuole. The reaction is sensitive to membrane dilution requires ATP and cytosol. Importantly cytosol made from a gene product. Materials and Methods Media Yeast strains were maintained on YPD media (1% yeast extract 2 peptone 2 dextrose and 2.5% bacto-agar). Liquid media for radiolabeling and plasmid maintenance was Wickerham’s minimal proline (WIMP) (Wickerham 1946) media supplemented with 0.5% yeast extract. Strains and Plasmids The yeast strains used in this study include BGY3300 (Gerhardt et al. 1998) gene under control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gene and a SalI site ～200 bp from the stop codon in pPRP33-100 which contains the complete gene in pBluescript KS (Stratagene Inc.). The resulting plasmid (pBG33BbSe) was digested with BamHI and SalI and subcloned into pGPD426 (Mumberg et al. 1995) to generate pGPD-BbSe-2. A six-histidine tag was placed at the NH2 terminus of with the PCR using 5′-TACGGATCCATGAGAGGATCGCATCACCATCACCATCACGGTTCTAGATTTTGGAATACTAAG-3′ as the forward primer and 5′-CAAAAAAGCTTGCCTTTGTTGCAAAG-3′ as the reverse primer. The amplicon was digested with BamHI and ClaI and subcloned into pGPD-BbSe-2 to generate pGPDHIS633-2. Antibody Production Two previously described fusion constructs (Banta et al. 1990) were expressed in and the insoluble fraction of cell lysates was prepared (Koerner et al. 1991). After SDS-PAGE the trpe-Vps33p fusion proteins were cut out of the gel and the gel slice used as antigens in rabbits at Cocalico Biologicals Inc. Antiserum against Vam3p was a generous gift of William Wickner (Dartmouth Medical School). A protein A-Sepharose column was used to purify total IgG from pre- and immune Vps33p rabbit sera. Preparation of Cytosol Yeast strain TVY614 was grown at 30°C in YPD (with 5% glucose) to an OD600 of 4-6 (usually 800-1 600 total OD600 units of cells were used). The cells were.