Synthesis of functional bovine opsin in insect cells under control of the baculovirus polyhedrin promoter

In vitro expression of cDNA encoding bovine opsin is accomplished using the baculovirus expression vector system. Full-length opsin was synthesized which was recognized by poly- and monoclonal antisera raised against bovine rhodopsin. Upon infection with a recombinant virus, 1×10⁶ insect cells produced up to 3 g opsin. Incubation of the in vitro synthesized opsin with 11-cis retinal produced a hydroxylamine-stable, photosensitive pigment.Abbreviations dpi days post infection - pfu plaque forming units - AcNPV Autographa californica nuclear polyhedrosis virus     

Practical Approaches to Low Density Lipoprotein Oxidation: Whys, Wherefores and Pitfalls

The purpose of this review is to bring together the different approaches for studying the oxidation of low density lipoproteins and try to identify some critical factors which will permit greater comparability between laboratories. These issues are discussed both in terms of the variety of exogenous mediators of oxidation applied (transition metal ions, haem proteins, azo initiators, peroxynitrite, cells etc.) and their raisons d'etre, as well as the methodologies (formation of conjugated dienes, hydroperoxides, decomposition products of lipid peroxidation, altered surface charge, macrophage uptake) applicable to the different stages of the oxidation and the factors underlying their accurate execution and interpretation.     

Sequences and Evolution of Human and Squirrel Monkey Blue Opsin Genes

The sequences of the entire blue opsin gene in the squirrel monkey (Saimiri boliviensis) and the five introns of the human blue opsin gene were obtained. Intron 3 of these genes contains an Alu sequence and intron 4 contains a partial mer13 sequence. A comparison of the squirrel monkey opsin sequence with published mammalian opsin sequences shows that features believed to be functionally critical are all conserved. However, the blue opsin has evolved twice as fast as rhodopsin and is only as conservative as the β globin, which has evolved at the average rate of mammalian proteins. Interestingly, the interhelical loops are, on average, actually more conservative than the transmembrane α helical regions. The introns of the blue opsin gene have evolved at the average rate of introns in primate genes. Received: 5 August 1996 / Accepted: 2 October 1996     

Effects of proteins on the thermotropic phase transitions of phospholipid membranes

A variety of proteins have been studied for their ability to interact and alter the thermotropic properties of phospholipid bilayer membranes as detected by differential scanning calorimeter. The proteins studied included: basic myelin protein (A1 protein), cytochrome c, major apoprotein of myelin proteolipid (N-2 apoprotein), gramicidin A, polylysine, ribonuclease and hemoglobin. The lipids used for the interactions were dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylglycerol. The interactions were grouped in three categories each having very different effects on the phospholipid phase transition from solid to liquid crystalline. The calorimetric studies were also correlated with data from vesicle permeability and monolayer expansion.Ribonuclease and polylysine which exemplify group 1 interactions, show strong dependence on electrostatic binding. Their effects on lipid bilayers include an increase in the enthalpy of transition (ΔH) accompanied by either an increase or no change in the temperature of transition (T_c). In addition, they show minimal effects on vesicle permeability and monolayer expansion. It was concluded that these interactions represent simple surface binding of the protein on the lipid bilayer without penetration into the hydrocarbon region.Cytochrome c and Al protein, which exemplify group 2 interactions, also show a strong dependence on the presence of net negative charges on the lipid bilayers for their binding. In contrast to the first group, however, they induce a drastic decrease in both T_c and ΔH of the lipid phase transition. Furthermore, they induce a large increase in the permeability of vesicles and a substantial expansion in area of closely packed monolayers at the air-water interface. It was concluded that group 2 interactions represent surface binding followed by partial penetration and/or deformation of the bilayer.Group 3 interactions, shown by proteolipid apoprotein and gramicidin A, were primarily non-polar in character, not requiring electrostatic charges and not inhibited by salt and pH changes. They had no appreciable effect on the T_c but did induce a linear decrease in the magnitude of the ΔH, proportional to the percentage of protein by weight. Membranes containing 50% proteolipid protein still exhibited a thermotropic transition with a ΔH one half that of the pure lipid, and only a small diminution of the size of the cooperative unit. It was concluded that in this case the protein was embedded within the bilayer, associating with a limited number of molecules via non-polar interactions, while the rest of the bilayer was largely unperturbed.     

Forthcoming Meetings 1993

The purpose of this review is to bring together the different approaches for studying the oxidation of low density lipoproteins and try to identify some critical factors which will permit greater comparability between laboratories. These issues are discussed both in terms of the variety of exogenous mediators of oxidation applied (transition metal ions, haem proteins, azo initiators, peroxynitrite, cells etc.) and their raisons d'etre, as well as the methodologies (formation of conjugated dienes, hydroperoxides, decomposition products of lipid peroxidation, altered surface charge, macrophage uptake) applicable to the different stages of the oxidation and the factors underlying their accurate execution and interpretation.     

Endoplasmic reticulum-associated degradation of a degron-containing polytopic membrane protein

Abstract

The presence of two basic amino acids strategically located within a single spanning transmembrane region has previously been shown to act as a signal for the endoplasmic reticulum associated degradation (ERAD) of several polypeptides. In contrast, the functionality of this degron motif within the context of a polytopic membrane protein has not been established. Using opsin as a model system, we have investigated the consequences of inserting the degron motif in the first of its seven transmembrane (TM) spans. Whilst these basic residue reduce the binding of the targeting factor, signal recognition particle, to the first TM span, this has no effect on membrane integration in vitro or in vivo. This most likely reflects the presence of multiple TM spans that can act as targeting signals within in the nascent opsin chain. We find that the degron motif leads to the efficient retention of mutant opsin chains at the endoplasmic reticulum. The mutant opsin polypeptides are degraded via a proteasomal pathway that involves the actions of the E3 ubiquitin ligase HRD1. In contrast, wild-type opsin remains stable for a prolonged period even when artificially accumulated at the endoplasmic reticulum. We conclude that a single dibasic degron motif is sufficient to initiate both the ER retention and subsequent degradation of ospin via an ERAD pathway.