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.