John K. Rose

Altered cytoplasmic domains affect intracellular transport of the vesicular stomatitis virus glycoprotein

We have altered the structure of the COOH-terminus of the vesicular stomatitis virus (VSV) glycoprotein (G) by introducing deletions into a cDNA clone encoding G protein. We examined the effects of these deletions on intracellular transport of G protein after expression of the deleted genes in eucaryotic cells under control of the SV40 late promoter. To prevent readthrough of translation into vector sequences, we introduced synthetic DNA linkers containing translation stop codons at the site of the deletion. G proteins that lacked the cytoplasmic domain and most of the transmembrane domain were secreted slowly from the cells. Deletion mutants affecting the structure of the cytoplasmic domain fell into two classes. The first class completely arrested transport of the protein to the cell surface at a stage prior to acquisition of complex oligosaccharides. The second class showed severely reduced rates of complex sugar addition although the proteins were eventually transported to the cell surface. Indirect immunofluorescence microscopy suggested that mutant proteins in both classes may accumulate in the rough endoplasmic reticulum.

Complete intergenic and flanking gene sequences from the genome of vesicular stomatitis virus

Abstract Plasmids containing vesicular stomatitis virus (VSV) mRNA sequences were used to obtain sequences corresponding to the 3′ termini of VSV mRNAs and to generate primers that were used to sequence through the genomic RNA regions joining the NS, M, G and L genes. Together with the sequence of the N-NS junction (McGeoch, 1979) these results provide the complete set of VSV intergenic and flanking gene sequences. Extensive homologies were found among the four junctions of the five VSV genes. These regions have the common structure (3′)AUACUUUUUUUNAUUGUCNNUA(5′), in which N indicates three variable positions in the 23 nucleotide sequence. The first eleven nucleotides of this sequence are complementary to the sequence (5′)…UAUGAAAAAA…(3′) which occurs at the mRNA-poly(A) junction in each mRNA. These sequences presumably signal polyadenylation of each mRNA. Dinucleotide spacers (CA or GA) whose complements do not appear in the mRNAs follow the polyadenylation signals and constitute the intergenic regions. Immediately after these dinucleotides are the sequences complementary to the 5′ terminal sequences on each mRNA, (5′)AACAGNNAUC(3′). Transcription events at these junctions, and the locations of possible promoter sequences preceding them, are discussed in terms of models of VSV transcription. Important features of VSV mRNA structure, including the location of a secondary ribosome binding site at an out-of-frame AUG codon in the N mRNA and the sequences of the M and G mRNAs preceding their ribosome binding sites, are also considered.

Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G) contains 20 uncharged and mostly hydrophobic amino acids. We created DNAs specifying G proteins with shortened transmembrane domains, by oligonucleotide-directed mutagenesis. Expression of these DNAs showed that G proteins containing 18, 16, or 14 amino acids of the original transmembrane domain assumed a transmembrane configuration and were transported to the cell surface. G proteins containing only 12 or 8 amino acids of this domain also spanned intracellular membranes, but their transport was blocked within a Golgi-like region in the cell. A G protein completely lacking the membrane-spanning domain accumulated in the endoplasmic reticulum and was secreted slowly. These experiments indicate that the size of the transmembrane domain is critical not only for membrane anchoring, but also for normal cell surface transport.

A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface.

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

Glycosylation allows cell-surface transport of an anchored secretory protein

We have previously described the construction and expression of a hybrid gene encoding a membrane-anchored form of rat growth hormone. This protein is anchored in cellular membranes by a carboxy-terminal extension composed of the transmembrane and cytoplasmic domains of the vesicular stomatitis virus glycoprotein. The protein is transported efficiently to the Golgi apparatus but not to the cell surface. To examine the possibility that N-linked glycosylation might be required for protein transport to the cell surface, we created two mutant proteins (using in vitro mutagenesis) in which single amino acids at two random sites in anchored growth hormone were changed to generate consensus sequences required for addition of N-linked carbohydrate. These mutant proteins, and a protein with both glycosylation sites, were glycosylated and were transported to the cell surface. These results suggest that N-linked glycosylation can serve as a signal for protein transport to the cell surface.

Conversion of a secretory protein into a transmembrane protein results in its transport to the golgi complex but not to the cell surface

We have carried out experiments designed to ask if it is possible to convert a secretory protein into an integral membrane protein by appending the membrane spanning domain of an integral membrane protein to its carboxy terminus. We first obtained expression of a cDNA clone encoding rat growth hormone (rGH) in eucaryotic cells, and found that this protein was secreted. We then constructed and expressed a hybrid gene encoding rGH fused to the membrane spanning and cytoplasmic domains of the vesicular stomatitis virus (VSV) glycoprotein (G). This fusion protein was anchored in microsomal membranes in the expected transmembrane configuration. The fusion protein was transported to the Golgi apparatus, and was esterified to palmitic acid, but it was not transported to the cell surface. We suggest that the sorting signal which allows rapid secretion of soluble rGH does not function when the protein is bound to the membrane.

ANALYSIS OF VSV GLYCOPROTEIN STRUCTURE AND GENOME STRUCTURE USING CLONED DNA

ABSTRACT We have determined the COOH-terminal and NH2-terminal sequences of the vesicular stomatitis virus (VSV) glycoprotein (G). The COOH-terminal sequence was deduced from the DNA sequence of a cloned DNA insert derived from the 3′-end of the G mRNA. The NH2-terminal sequence was deduced from the sequence of a DNA primer extended on the VSV genome from the M gene into the adjacent G gene. We have shown that an uninterrupted hydrophobic domain near the COOH-terminus of G spans the lipid bilayer and that the highly basic COOH-terminus resides inside the virion. The functional significance of both the COOH-terminal and NH2-terminal sequences is discussed. The sequences in the VSV genome at the junctions of the NS-M, M-G, and G-L genes were determined using DNA primers derived from cDNA clones. The junctions have the common sequence (3′) AUACUUUUUUU NA UUGUCNNUAG (5′) in which the underlined dinucleotide is the intergenic region. The sequence preceding this dinucleotide is complementary to each mRNA at the site of polyadenylation, and the sequence following the dinucleotide is complementary to the capped 5′-terminal sequence of each mRNA. Polyadenylation by repetitive copying of the 7 U residues and other possible transcription events at these junctions are discussed.