Cytoplasmic domains of cellular and viral integral membrane proteins substitute for the cytoplasmic domain of the vesicular stomatitis virus glycoprotein in transport to the plasma membrane.

Abstract

Abstract. Oligonucleotide-directed mutagenesis was used to construct chimeric cDNAs that encode the extracellular and transmembrane domains of the vesicular stomatitis virus glycoprotein (G) linked to the cytoplasmic domain of either the immunoglobulin u membrane heavy chain, the hemagglutinin glycoprotein of influenza virus, or the small glycoprotein (023) of infectious bronchitis virus. Biochemical analyses and immunofluorescence microscopy demonstrated that these hybrid genes were correctly expressed in eukaryotic cells and that the hybrid proteins were transported to the plasma membrane. The rate of transport to the Golgi complex of G protein with an immunoglobulin # membrane cytoplasmic domain was approximately sixfold slower than G protein with its normal cytoplasmic domain. However, this rate was virtually identical to the rate of transport of ~m heavy chain molecules measured in the B cell line WEHI 231. The rate of transport of G protein with a hemagglutinin cytoplasmic domain was threefold slower than wild type G protein and G protein with a p23 cytoplasmic domain, which were transported at similar rates. The combined results underscore the importance of the amino acid sequence in the cytoplasmic domain for efficient transport of G protein to the cell surface. Also, normal cytoplasmic domains from other transmembrane glycoproteins can substitute for the G protein cytoplasmic domain in transport of G protein to the plasma membrane. The method of constructing precise hybrid proteins described here will be useful in defining functions of specific domains of viral and cellular integral membrane proteins.

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

“…2). Mutant 1473 is completely inhibited in transport, only 10% of TMS is transported after 2 h, and GIx, TMR, and GHA are transported efficiently but four-to eightfold more slowly than wt G protein (Puddington et al, 1986). The results showed that trimerization alone is not sufficient for transport.…”

“…This group of mutants had alterations in the cytoplasmic domain. Mutant 1473 was completely blocked in transport, only 10% of TMS was transported after 2 h, while GHA, G~t, and TMR were transported efficiently but at one-fourth to one-eighth the wt rate (Puddington et al, 1986). The mutants acquired all the antigenic epitopes characteristic of the mature G protein and, with the exception of TMS, they trimerized with wt kinetics.…”

“…This difference suggested that the ectodomain folds independently of the COOH-terminal domain. Indeed, both G and HA mutants are rather tolerant to changes in both their cytoplasmic and transmembrane domains (Adams and Rose, 1985a, b;Doyle et al, 1985Doyle et al, , 1986Roth et al, 1986;Puddington et al, 1986). While these regions may be important for the final stability and transport of the G and HA oligomers , they do not seem to play important roles in initiating trimer formation.…”

“…Each of the mutant proteins used in this study had point mutations, deletions, or insertions in one of these domains. Their transport phenotypes have been previously described and found to range from near normal transport to the cell surface to partial or complete inhibition (Rose and Bergmann, 1983;Adams and Rose, 1985b;Gabel and Bergmann, 1985;Gallione and Rose, 1985;Puddington et al, 1986;Scullion et al, 1987;Machamer and Rose, 1988a, b). Table I contains a summary of the mutants, the amino acid sequence changes, and their transport phenotypes.…”

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

“…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 protein and not directly related to interactions on the cytoplasmic side.…”