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Abstract. We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the 1gp120 (lgp--A) to lysosomes . Using several surface labeling and internalization assays, our results suggest that 1gp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors : expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type 1gp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4°C. With increased expression levels, however, an increased fraction of trans-YSOSOMES contain a characteristic set of highly glycosylated membrane proteins. By cloning cDNAs from different species (Cha et al

Peroxisomal membrane protein (Pmp)26p (RnPex11p), a major constituent of induced rat liver peroxisomal membrane, was found to contain a COOH-terminal, cytoplasmically exposed consensus dilysine motif with the potential to bind coatomer. Biochemical as well as immunocytochemical evidence is presented showing that peroxisomes incubated with preparations of bovine brain or rat liver cytosol recruit ADP-ribosylation factor (ARF) and coatomer in a strictly guanosine 5′-O-(3-thiotriphosphate)–dependent manner. Consistent with this observation, ldlF cells expressing a temperature-sensitive mutant version of the ε-subunit of coatomer exhibit elongated tubular peroxisomes possibly due to impaired vesiculation at the nonpermissive temperature. Since overexpression of Pex11p in Chinese hamster ovary wild-type cells causes proliferation of peroxisomes, these data suggest that Pex11p plays an important role in peroxisome biogenesis by supporting ARF- and coatomer-dependent vesiculation of the organelles.

A site-directed photocrosslink approach was used to elucidate components that interact directly with ADPribosylation factor (ARF)-GTP during coat assembly. Two ARF mutants were generated that contain a photolabile amino acid at positions distant to each other within the ARF molecule. Here we show that one of the two positions specifically interacts with coatomer subunit ␤ both on Golgi membranes and in isolated coat protein complex type I (COPI)-coated vesicles. Thus, a direct and GTP-dependent interaction of coatomer via ␤-coat protein complex (COP) with ARF is involved in the coating of COPI-coated vesicles. These data implicate a bivalent interaction of the complex with the donor membrane during vesicle formation.

Abstract. Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] a-X), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine a-COP, the 135-kD subunit of coatomer as well as ~-COP, the 57-kD subunit and have identified a yeast homolog of 8-COP by cDNA sequence comparison and by NHz-terminal peptide sequencing. ~-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the 8-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between a-and ~-COPs and between 13-and ~-COPs. Moreover, the two-hybrid system indicates interactions between -,/-and E-COPs as well as between c~-and [Y-COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.