The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER.
Abstract. We report an essential role for the ras-
Abstract. Members of the rab/YPTI/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocyfic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rabla mutant, rabla(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rabl and B-COP, a subunit of the coatomer complex. Similar to previous observations . Cell. 76:841-852), VSV-G was concentrated nearly 5-1G-fold in vesicular carriers that aocumulate in the presence of the rabla(N124I) mutant. VSV-G containing vesicles and vesiculartubular clusters were also found to accumulate in the presence of a rabla effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca 2+. These results suggest that the combined action of a Ca2+-dependent prorein and conformational changes associated with the GTPase cycle of rabl are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.
Abstract. The small GTPase Rabl is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rabl in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (h/2 < 30 s) extracted Rabl from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and transGolgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rabl fractionates with a molecular mass of ,,o80 kD in the form of a GDI-Rabl complex. When the GDI-Rabl complex was depleted from cytosol by use of a Rabl-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rabl complex prepared in vitro from recombinant forms of Rabl and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rabl complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.
Synthetic peptides of the putative effector domain of members of the ras‐related rab gene family of small GTP‐binding proteins were synthesized and found to be potent inhibitors of endoplasmic reticulum (ER) to Golgi and intra‐Golgi transport in vitro. Inhibition of transport by one of the effector domain peptides was rapid (t1/2 of 30 s), and irreversible. Analysis of the temporal site of peptide inhibition indicated that a late step in transport was blocked, coincident with a Ca2(+)‐dependent prefusion step. The results provide novel biochemical evidence for the role of members of the rab gene family in vesicular transport in mammalian cells, and implicate a role for a new downstream Rab effector protein (REP) regulating vesicle fusion.
In addition to diacyl glycerophosphocholine and sphingomyelin, human plasma also contains small amounts of other glycerophospholipids, which may have special metabolic function. The structure and origin of these minor plasma lipids has not been determined. Knowledge of the detailed composition of the phospholipids of red blood cells (Myher et al., Lipids 24, 1989) permits evaluation of one of the possible sources. This study reports the detailed analyses of plasma glycerophospholipids made in parallel to those of the erythrocyte lipids obtained from the same blood using HPLC and GLC methods. The proportions of the major phospholipid classes in the plasma and erythrocytes were similar to published values, including the essential absence of diradyl glycerophosphoserine from plasma. Plasma diradyl glycerophosphocholine contained 93.0% diacyl, 3.4% alkylkacyl and 3.6% alkenylacyl, whereas the diradyl glycerophosphoethanolamine consisted of 71.8% alkenylacyl, 19.9% diacyl and 8.3% alkylacyl subclasses. The diradyl glycerophosphoinositol was 100% diacyl. The content of the minor subclasses of plasma diradyl glycerophosphocholine is similar to that of the red cells, but the ether content of the diradyl glycerophosphoethanolamine is higher in plasma than in cells. The lipid ether subclasses of plasma glycerophospholipids also contained a higher proportion of the C20, C22 and C24 alkyl and alkenyl chains than those of the cells. Furthermore, the C16 and C18-containing species in diradyl glycerophosphoethanolamine subclasses varied with the nature of the polyunsaturated acid, whereas in diradyl glycerophosphocholine subclasses the polyunsaturated acids were combined with the C16 and C18 acids in equal proportions. The significant differences in the molecular species of glycerophospholipids and sphingomyelin between plasma and red cells would appear to limit any direct transfer or equilibration of their lipid components.
This study reports the application of modern methods of molecular species analysis in determination of the structure of both major and minor glycerophospholipids and sphingomyelins of human erythrocytes. Individual phospholipid classes were resolved from total lipid extracts by thin-layer chromatography. Diradylglycerols were released by phospholipase C and converted into trimethylsilyl ethers, which were resolved into the alkenylacyl, alkylacyl and diacylglycerol subclasses by normal phase high performance liquid chromatography. Molecular species of diradylglycerols and ceramides were quantitated according to carbon and double bond number by gas liquid chromatography using a fused silica capillary column wall-coated with bonded RTx-2330. The molecular species of ceramides were determined by GC/MS. The diradyl glycerophosphocholines contained 93.0% diacyl, 4.6% alkylacyl and 2.5% alkenylacyl, while the diradyl glycerophosphoethanolamines were made up of 48.8% diacyl, 47.8% alkenylacyl and 3.4% alkylacyl subclasses. Analysis of the molecular species showed that the long chain polyunsaturated acids were mainly combined with C16 in all diradyl GPC subclasses and in diacyl GPE, while in the alkylacyl and alkenylacyl GPE and in diacyl glycerophosphoinositol and diacyl glycerophosphoserine they were combined mainly with C18 saturated fatty chains. In addition to the C16 and C18 alkyl and alkenyl, the ether fractions also contained significant proportions of C20, C22 and C24 chains. The molecular species of the ceramide moieties of the SPH were made up largely of mono- and diunsaturated species. Over 200 molecular species were identified and quantitated in a representative sample of human red blood cells.
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