Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta, gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.

Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, occurs in the majority of renal clear cell carcinomas (RCC). VHL's function, regulating the degradation of hypoxia-inducible factor A (HIFA) subunits, explains the angiogenic nature of these tumors, but not tumor initiation. Because the development of renal cysts precedes tumor formation, and because the dysfunction of primary cilium is a common pathogenic mechanism in polycystic kidney diseases, we determined whether kidney-derived VHLÀ cells required VHL for the generation of cilium. Ectopic expression of VHL in RCC(VHLÀ) cells induced increased polarization and primary cilium formation. Cilium formation correlated directly with the expression of both wild-type VHL isoforms and a VHL mutant not associated with RCC development, whereas expression of RCC-associated VHL mutants did not support ciliogenesis. Requirement of VHL for ciliogenesis was independent of HIFA abundance. These data indicate separable independent functions for VHL (HIFA degradation and differentiation) and suggest a mechanism whereby disruption of both functions is required for renal carcinogenesis. (Cancer Res 2006; 66(14): 6903-7)

High-gradient magnetic affinity chromatography (HDMAC) has been used to obtain highly enriched plasma membranes, free of intracellular membrane contaminmts, from cultured Chinese hamster ovary (CHO) cells in yields of .80%. Using this procedure we have characterized the transport of glucosYceramlde (GlcCer) and the gangoide GM3 to the plasma membrane. Newly synthesized GlcCer reaches the plasma membrane in 7.2 min, whereas GM3 requires 21.5 min to reach the plasma membrane. Brefeldin A prevents transport of newly synthesized GM3 and sWhinmyelmn to the plasma membrane but has no effect on the transport of GIcCer. Similarly, incubation of CHO cells at 15°C blocks transport of GM3 and sphingomyelin to the plasma membrane but has no effect on GicCer movement. We propose that carrier-mediated transport accounts for a major fraction of the plasma membrane GlcCer. Puls-chase studies with either [3Hglcose or [3pH] (2) and ganglioside GM3 (3) as well as short-chain derivatives of sphingomyelin and glucosylceramide (GlcCer) (4-6) to the plasma membrane. (ii) Soluble carriers such as lipid-transfer proteins account for a major portion of the movement of the phospholipids phosphatidylcholine (7) and phosphatidylethanolamine (8, 9) to the plasma membrane. (iii) A vesicular transport pathway which is distinct from the secretory pathway accounts for the transport of newly synthesized cholesterol (10) to the plasma membrane. Transfer ofglucose to ceramide by UDPglucoseceramide glucosyltransferase has been localized to the cytosolic surface of the Golgi apparatus (11)(12)(13)(14), indicating that GlcCer must be translocated to the luminal leaflet for higher glycosylation to occur. We have determined that 45% of GlcCer in Chinese hamster ovary (CHO) cells is located in the plasma membrane (15). Since GlcCer is synthesized on the cytosolic surface of the Golgi membrane and translocated to the luminal surface, GlcCer could be transported to the plasma membrane through the vesicular protein secretory pathway and/or by an alternative pathway through the cytosol, perhaps by lipid-transfer proteins as proposed by Sasaki (16 (vol/vol) heat-inactivated fetal bovine serum. CHO K1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10%6 iron-supplemented heat-inactivated fetal bovine serum, 2 mM glutamine, 1x nonessential amino acids, 1 mM proline, and lx penicillin/streptomycin. Cells were released by trypsin/EDTA treatment and plated on fresh culture dishes 48 hr prior to experimental manipulation.Metabolic Labeling. For pulse-chase experiments, cells were rinsed three times in Dulbecco's phosphate-buffered saline (PBS) and incubated for 1 hr at 370C in methionine-and cysteine-free medium. The medium was replaced with fresh methionine-and cysteine-free medium containing Tran35S-label at 150 pCi/ml. After 20 min of-incubation, incorporation was terminated by washing twice with PBS and the chase was initiated by addition of complete medium. For metabolic labeling of lipids [3H]palmitate [10 puCi/ml ...

Brefeldin A (BFA) induces the rapid redistribution of the Golgi complex into the endoplasmic reticulum (ER), causing the glycoproteins that are retained in the ER to be processed by Golgi enzymes. We have examined the effects of BFA on the synthesis of glycosphingolipids (GSL) to map the intracellular sites of GSL synthesis. In several cultured cell

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