Milton Adesnik

Genes for cytochrome P-450 and their regulation

The capacity of the liver microsomal mixed-function oxidase system to metabolize a wide variety of exogenous as well as endogenous compounds reflects the participation of multiple forms of the terminal oxidase, cytochrome P-450, which have different broad, but overlapping, substrate specificities. Several of these isozymes accumulate in the liver after exposure of animals to specific inducing agents. Recent studies employing recombinant DNA techniques to investigate the genetic and evolutionary relatedness of various cytochrome P-450 isozymes as well as the molecular basis for the induction phenomenon are described. The conclusions from these investigations are presented in the context of the substantial body of data obtained from the characterization of specific cytochrome P-450 isozymes and from studies on the induction of specific isozymes or enzymatic activities during development or after treatment of animals with various inducing agents.

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.

Biosynthesis of hepatocyte endoplasmic reticulum proteins

Publisher Summary This chapter explores the application of rat hepatocyte that can serve as an excellent model for the study of the biosynthesis of endoplasmic reticulum (ER) membrane proteins. In the hepatocyte, as in all cell types, the biosynthetic machinery for the synthesis of secretory and membrane proteins includes both rough and smooth ER membrane-associated components, which affect a variety of co- and post-translational modifications of polypeptides synthesized in bound polysomes. Certain integral membrane proteins, such as cytochrome b 5 and NADH-cytochrome b 5 reductase, which are anchored to the membranes solely by a small carboxy terminal segment, are incorporated post-translationally into the ER membrane after their discharge from free polysomes into the cell sap. Other ER membrane proteins, however, only two of which have been demonstrated to be glycoproteins and to span the membrane, are synthesized in membrane-bound ribosomes and are inserted into the membrane during their synthesis. The fact that the concentrations of cytochrome b 5 and its reductase are much higher in the ER than in other membranes may reflect the affinity of these proteins for other components of the microsomal electron transport chains, such as those that are synthesized on bound polysomes.

Structural and Functional Aspects of the Protein Synthesizing Apparatus in the Rough Endoplasmic Reticulum

The successful implementation of a genetic program requires that upon completion of translation polypeptides released from ribosomes are transferred to their final destination in the cell This appears to be a rather formidable process since, in a eukaryotic cell, the fate of a protein can be at least as diverse as the membranous structures within the cell and the subcellular compartments which they separate. For many proteins, these subcellular compartments serve only as the initial destination; transfer to another compartment or export from the cell may be their ultimate fate.

Christian de Duve: Explorer of the cell who discovered new organelles by using a centrifuge

Christian de Duve, whose laboratory in Louvain discovered lysosomes in 1955 and defined peroxisomes in 1965, died at his home in Nethen, Belgium at the age of 95, on May 4, 2013. De Duve was the last of a group of eminent physiological chemists who, by the 1940s and 1950s, began to explore the subcellular organization of biochemical pathways and thus forged the emergence of Modern Cell Biology. Christian De Duve, Albert Claude, and George Palade received the Nobel Prize in 1974 “for their discoveries concerning the structural and functional organization of the cell.”

David Sabatini – a lifelong fascination with organelles

As a pioneer molecular cell biologist, highly skilled in both morphological and biochemical approaches, David Sabatini was a key figure in laying the foundation for the field of intracellular protein trafficking with his seminal studies on cotranslational translocation of nascent polypeptides in the endoplasmic reticulum and the intracellular sorting of plasma membrane proteins in polarized epithelial cells.