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Abstract. In the companion paper (Huber, L. A.,

For identification of Rab, Rac, Rho, Ral, Rap, and Arfproteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to highresolution two-dimensional polyacrylamide gel electrophoresis followed by [a-32P] (17,19). Primary cultures of rat hippocampal neurons were prepared as described (20). Cells were cultured for 9 days (stage 5 cells) before use (21).Infection with T7 polymerase-recombinant vaccinia virus (22) was carried out in serum-free medium (17). After 30 min cells were supplemented with 5% serum and transfected by using N- [1-(2,3-dioleoyloxy)propyl]-N,NN-trimethylammonium methyl sulfate (DOTAP) (Boehringer Mannheim) according to the manufacturer's instructions and incubated in the presence of 10 mM hydroxyurea for 5 hr at 370C in a 5% CO2 atmosphere.Preparation of Total Cell Membrane Fractions and Synaptosomes. A postnuclear supernatant (17) was transferred to a Beckman TLA100.2 microcentrifuge tube and centrifuged at 60,000 rpm for 30 min at 40C. The supernatant (cytosol fraction) and the pellet were separated and processed for analysis. A fraction enriched in synaptosomes (18) was isolated from 15 rat brains. Two-Dimensional PAGE and Transfer to Nitrocellulose for [e-32P]GTP Overlay and Immunoblotting. A combination of isoelectric focusing (IEF) and SDS/PAGE was used to resolve proteins in two dimensions essentially as described previously (17). For transfer to nitrocellulose for GTP overlay and immunoblotting we have modified a protocol (17) Abbreviations: IEF, isoelectric focusing; GDI, GDP dissociation inhibitor; GST, glutathione S-transferase. tPresent address:

The induction kinetics and surface accessibility of the outer membrane lipoprotein were studied in an Escherichia coli strain with the Ipp Thus, topological studies of lipoprotein distribution in the outer membrane and of lipoprotein attachment to the peptidoglycan layer might be useful in improving our understanding of cell wall morphology and biosynthesis. For such studies (29), it is necessary to regulate the lipoprotein levels of the cell and to detect the newly inserted and linked molecules by immunoelectron microscopy.In this paper we approach both of these problems. The first condition was met by using E. coli JA221 Ipp/F' lacIP(pKEN125), which has a deletion in the chromosomal Ipp gene, whereas plasmid pKEN125 encodes lipoprotein under lacUV5 promoter control (21). The MATERIALS AND METHODS Organisms, growth, and radiolabeling conditions. E. coli JA221 lppIF' lacIq and E. coli JA221 IppIF' lacIP(pKEN125) were grown on M9 medium (20) supplemented with Ltryptophan (100 pug/ml) and L-leucine (100 ,ug/ml), with 0.5% (wt/vol) glucose or 0.5% (wt/vol) lactose as the carbon source. The strain harboring plasmid pKEN125 was grown in the presence of ampicillin (50 p.g/ml). E. coli JE5513, which lacks lipoprotein (5), was grown on Vogel and Bonner E salts medium (27) with 0.5% (wt/vol) glucose as the carbon source. All strains were grown with shaking (200 rpm) at 37°C. Cells containing plasmid pKEN125 growing exponentially on glucose were induced for lipoprotein with 2 mM IPTG (isopropyl-3-D-thiogalactopyranoside; Calbiochem-