Ivan E. Ivanov

Endomembrane Trafficking of Ras: The CAAX Motif Targets Proteins to the ER and Golgi

We show that Nras is transiently localized in the Golgi prior to the plasma membrane (PM). Moreover, green fluorescent protein (GFP)-tagged Nras illuminated motile, peri-Golgi vesicles, and prolonged BFA treatment blocked PM expression. GFP-Hras colocalized with GFP-Nras, but GFP-Kras4B revealed less Golgi and no vesicular fluorescence. Whereas a secondary membrane targeting signal was required for PM expression, the CAAX motif alone was necessary and sufficient to target proteins to the endomembrane where they were methylated, a modification required for efficient membrane association. Thus, prenylated CAAX proteins do not associate directly with the PM but instead associate with the endomembrane and are subsequently transported to the PM, a process that requires a secondary targeting motif.

The in Vitro Generation of Post-Golgi Vesicles Carrying Viral Envelope Glycoproteins Requires an ARF-like GTP-binding Protein and a Protein Kinase C Associated with the Golgi Apparatus*

We have developed a system that recreates in vitro the generation of post-Golgi vesicles from an isolated Golgi fraction prepared from vesicular stomatitis virus- or influenza virus-infected Madin-Darby canine kidney or HepG2 cells. In this system, vesicle generation is temperature- and ATP-dependent and requires a supply of cytosolic proteins, including an N-ethylmaleimide-sensitive factor distinct from NSF. Cytosolic proteins obtained from yeast were as effective as mammalian cytosolic proteins in supporting vesicle formation and had the same requirements. The vesicles produced (50-80 nm in diameter) are depleted of the trans Golgi marker sialyltransferase, contain the viral glycoprotein molecules with their cytoplasmic tails exposed, and do not show an easily recognizable protein coat. Vesicle generation was inhibited by brefeldin A, which indicates that it requires the activation of an Arf-like GTP-binding protein that promotes assembly of a vesicle coat. Vesicles formed in the presence of the nonhydrolyzable GTP analogue guanosine 5′-3-O-(thio)triphosphate retained a nonclathrin protein coat resembling that of COP-coated vesicles, and sedimented more rapidly in a sucrose gradient than the uncoated ones generated in its absence. This indicates that GTP hydrolysis is not required for vesicle generation but that it is for vesicle uncoating. The activity of a Golgi-associated protein kinase C (PKC) was found to be necessary for the release of post-Golgi vesicles, as indicated by the capacity of a variety of inhibitors and antibodies to PKC to suppress it, as well as by the stimulatory effect of the PKC activator 12-O-tetradecanoylphorbol-13-acetate.

Surface features and handedness of a model for the eukaryotic small ribosomal subunit

A three-dimensional model with the appropriate handedness for the small (40 S) eukaryotic ribosomal subunit has been derived from electron microscopic observations of: (a) profiles of negatively stained particles and changes caused by tilting experiments, and (b) surface features of the subunits revealed by metal shadowing. The 40 S subunit is an elongated (270 × 180 × 160A) asymmetric and slightly bent particle which consists of two main regions separated by a circumaxial groove. The upper one-third is oval-shaped and the lower two-thirds has a wedge-shaped bottom portion. A prominence projects upward from the central portion of the subunit. A direct comparison between the features of eukaryotic rat liver 40 S and prokaryotic Escherichia coli 30 S subunits shows similarities in the central and upper one-third portions since main differences in overall size and shape seem primarily confined to the bottom portions of the subunits. In our previous work ( I. Emanuilov, D. Sabatini, J. A. Lake, and C. Freienstein, 1978 , Proc. Nat. Acad. Sci. USA 75 , 1389–1393) the site of attachment of the eukaryotic initiation factor 3 (eIF-3) to the native rabbit reticulocyte 40 S ribosomal subunit was localized on the back side of the central portion of these particles covering part of the large prominence. The same region of the 40 S ribosomal subunit appears to face the 60 S subunit in active monomeric ribosomes and thus is likely to play a functional role in the initiation of protein synthesis.