Effects of altered cytoplasmic domains on transport of the vesicular stomatitis virus glycoprotein are transferable to other proteins.

Abstract

Alterations of the cytoplasmic domain of the vesicular stomatitis virus glycoprotein (G protein) were shown previously to affect transport of the protein from the endoplasmic reticulum, and recent studies have shown that this occurs without detectable effects on G protein folding and trimerization (R. W. Doms et al., J. Cell Biol., in press). Deletions within this domain slowed exit of the mutant proteins from the endoplasmic reticulum, and replacement of this domain with a foreign 12-amino-acid sequence blocked all transport out of the endoplasmic reticulum. To extend these studies, we determined whether such effects of cytoplasmic domain changes were transferable to other proteins. Three different assays showed that the effects of the mutations on transport of two membrane-anchored secretary proteins were the same as those observed with vesicular stomatitis virus G protein. In addition, possible effects on oligomerization were examined for both transported and nontransported forms of membrane-anchored human chorionic gonadotropin-a. These membraneanchored forms, like the nonanchored human chorionic gonadotropin-a, had sedimentation coefficients consistent with a monomeric structure. Taken together, our results provide strong evidence that these cytoplasmic mutations affect transport by affecting interactions at or near the cytoplasmic side of the membrane.The vesicular stomatitis virus (VSV) glycoprotein (G protein) is a model for a major class of eucaryotic viral and cellular plasma membrane proteins. These proteins are synthesized on membrane-bound ribosomes, inserted into the lumen of the endoplasmic reticulum (ER) as nascent chains, and anchored via C-terminal hydrophobic sequences. Insertion of G protein is halted by a typical hydrophobic sequence of 20 amino acids followed by a short, C-terminal cytoplasmic domain of 29 amino acids (9, 19). Subsequent transport of membrane proteins such as G protein to the plasma membrane is thought to occur via vesicular carriers that bud from the ER. Lodish et al. (13) have made progress in identifying these carriers, but they are not yet well characterized. It is clear that both membrane and secretary proteins exit the ER at different rates, and these variations may reflect different rates of protein incorporation into transport vesicles (4, 12).Cytoplasmic domains of transmembrane proteins are clearly in a position where they might influence incorporation of the proteins into vesicular carriers. Several studies have assessed the role of cytoplasmic domains in transport of proteins from the ER to the cell surface. In these studies, deletions or substitutions of amino acids were made in cytoplasmic domains of several proteins. In some cases dramatic decreases in rates of transport or blocks in transport were observed, while in other cases the effect was minimal (2,11,18,20,21,25). One simple way to reconcile all of these results is to say that when an effect on transport is observed, it is due to an indirect effect on oligomerization or folding of the ectodomain of the...

Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains

“…For intracellular localization, the fixed cells were permeabilized with 1% Triton X-100 in PBS prior to incubation with the anti-G antibody. For quantitation of G protein expressed in the cell surface, lactoperoxidase-catalyzed iodination was done according to Guan et al (13). Relative amounts of G protein on the cell surface were determined by scanning densitometry of the fluorograms of the SDS-polyacrylamide gels.…”

Differential effects of mutations in three domains on folding, quaternary structure, and intracellular transport of vesicular stomatitis virus G protein.

“…The cytoplasmic sequence of the mutant 1473 also appears to serve as a retention signal, perhaps by interacting with cytoplasmic proteins or cytoplasmic domains of ER proteins. Indeed, when this sequence is placed on the cytoplasmic side of other membrane proteins, they too are retained in the ER (Guan et al, 1988).…”

Single amino acid substitutions in the glycoprotein B carboxy terminus influence the fusion from without property of herpes simplex virus type 1

“…Multiple mechanisms could be involved in the process by which FFWO activity is enhanced by amino acid alterations in the gB-1 C-t. A hypothesis of Guan et al (1988) for vesicular stomatitis virus G protein and Huff et al (1991) for HSV gB-1 predicts that the cytoplasmic part of these proteins is important for correct glycoprotein transport to the plasma membrane, including the correct formation of homodimers. In order to be certain that FFWO is a genuine effect of the mutated gB-1 and not an effect of higher amounts of gB-1 present on the surface of FFWO + viruses we developed an ELISA.…”