In-frame ATG Codon Research Articles

A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana.

To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene. The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels. Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable. The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings. Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae. The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening. This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made. HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes. We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

Type XIII Collagen Is Identified as a Plasma Membrane Protein

The complete primary structure of the mouse type XIII collagen chain was determined by cDNA cloning. Comparison of the mouse amino acid sequences with the previously determined human sequences revealed a high identity of 90%. Surprisingly, the mouse cDNAs extended further in the 5' direction than the previously identified human clones. The 5' sequences contained a new in-frame ATG codon for translation initiation which resulted in elongation of the N-terminal noncollagenous domain by 81 residues. These N-terminal sequences lack a typical signal sequence but include a highly hydrophobic segment that clearly fulfills the criteria for a transmembrane domain. The sequence data thus unexpectedly suggested that type XIII collagen may be located on the plasma membrane, with a short cytosolic N-terminal portion and a long collagenous extracellular portion. These sequence data prompted us to generate antipeptide antibodies against type XIII collagen in order to study the protein and its subcellular location. Western blotting of human tumor HT-1080 cell extract revealed bands of over 180 kDa. These appeared to represent disulfide-bonded multimeric polypeptide forms that resolved upon reduction into 85-95-kDa bands that are likely to represent a mixture of splice forms of monomeric type XIII collagen chains. These chains were shown to contain the predicted N-terminal extension and thus also the putative transmembrane segment. Immunoprecipitation of biotinylated type XIII collagen from surface-labeled HT-1080 cells, subcellular fractionation, and immunofluorescence staining were used to demonstrate that type XIII collagen molecules are indeed located in the plasma membranes of these cells.

New 5′ Regions of the Murine and Human Genes for DNA (Cytosine-5)-methyltransferase

DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5'-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5' sequences and the intron-exon structure of the 5' region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.

Molecular cloning of the three base restriction endonuclease R.CviJI from eukaryotic Chlorella virus IL-3A

R.CviJI is unique among site-specific restriction endonucleases in that its activity can be modulated to recognize either a two or three base sequence. Normally R.CviJI cleaves RGCY sites between the G and C to leave blunt ends. In the presence of ATP R.CviJI* cleaves RGCN and YGCY sites, but not YGCR sites. The gene encoding R.CviJI was cloned from the eukaryotic Chlorella virus IL-3A and expressed in Escherichia coli. The primary E.coli cviJIR gene product is a 278 amino acid protein initiated from a GTG codon, rather than the expected 358 amino acid protein initiated from an in-frame upstream ATG codon. Interestingly, the 278 amino acid protein displays the normal restriction activity but not the R.CviJI* activity of the native enzyme. Nine restriction and modification proteins which recognize a central GC or CG sequence share short regions of identity with R.CviJI amino acids 144-235, suggesting that this region is the recognition and/or catalytic domain.

The Plastid-encoded ccsA Gene Is Required for Heme Attachment to Chloroplast c-type Cytochromes

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Characterization of Sequences Controlling the Synthesis of Alfalfa Mosaic Virus Subgenomic RNA in Vivo

RNA 3 of alfalfa mosaic virus encodes the movement protein P3 and the vital coat protein (CP) CP is translated from a subgenomic (sg) messenger, RNA 4. To characterize the sg promoter that is responsible for RNA 4 synthesis in vivo, putative sg promoter sequences were inserted in a unique Xhol site located between the initiation codon of the P3 gene and a second in-frame ATG codon in an infectious cDNA clone of RNA 3. Mutants with an active sg promoter insert expressed an N-terminally truncated P3 protein and were able to accumulate in plants, In addition, sg promoter activity was analyzed in protoplasts. When the transcription start site is taken as +1, the sequence of nucleotides -26/+1 was found to have a basal level of sg promoter activity, This activity was increased to near maximum levels when the sg promoter sequence was extended to -136/+12. The upstream positive regulatory element was mapped to nucleotides -136/-94. Engineering of point mutations and small deletions in RNA 3 around the transcription start site for RNA 4 synthesis revealed elements important for sg promoter activity with similarity to sequences conserved in sg promoters of alpha-like viruses. Some of these elements appeared to be required in cis for RNA 3 accumulation. A deletion of the C-terminal three amino acids of the P3 protein rendered this protein nonfunctional in cell-to-cell movement.

Novel short transcripts of hepatitis B virus X gene derived from intragenic promoter.

The hepatitis B virus X gene encodes a transcriptional trans-activator that appears essential for viral replication in the infected liver. It has been shown by others that at least two X protein products, initiating at different in-frame ATG codons within the X open reading frame, are necessary for full trans-activating function. Yet, previous studies have demonstrated only a full-length X gene transcript that extends 5' of the first ATG codon in the X open reading frame. In this communication, we present evidence for the existence of shorter X gene transcripts, whose heterogeneous 5' ends straddle the second ATG codon of the X open reading frame. These transcripts are not degradation products of the full-length X gene transcripts but rather arise from an independent promoter. Furthermore, immunofluorescence and functional analyses reveal that active X proteins are translated from these transcripts. Therefore, there appears to be a previously undescribed promoter embedded within the X open reading frame that can give rise to functionally important transcripts.

Expression, functional analysis, and in situ hybridization of a cloned rat kidney collecting duct water channel

The cloning and expression of an apical membrane water channel from rat kidney collecting duct (WCH-CD) homologous to a 28-kDa integral membrane protein (CHIP28) was reported recently (K. Fushimi, S. Uchida, Y. Hara, Y. Hirata, F. Marumo, and S. Sasaki. Nature Lond. 361: 549-552, 1993). We obtained an approximately 1.8-kilobase clone from a rat kidney lambda gt10 cDNA library by a polymerase chain reaction cloning method; whereas the coding sequence (814 base pairs, predicted protein size 29 kDa) was identical to that reported, we identified an in-frame ATG codon at base pair -123 predicting a protein size of 33 kDa. On Northern blots probed by cDNAs corresponding to the WCH-CD coding sequence (base pairs +1 to +814) or 5'-untranslated sequence (-403 to -16), a single band at 1.9 kilobases was observed in kidney medulla greater than in cortex but not in other tissues; mRNA expression was increased strongly by dehydration. Translation and oocyte expression studies were performed to identify the translation start site. The short (base pairs +1 to +814) and long (base pairs -123 to +814) cDNAs were subcloned in vector pSP64 containing the 5'-untranslated Xenopus globin sequence upstream to the ATGs; a 30-base pair c-myc sequence was engineered at the COOH- terminal for antibody recognition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of X Protein on Transactivation of Hepatitis B Virus Promoters and on Viral Replication

The X gene product of hepatitis B virus (HBV) transactivates a wide variety of promoters, including four promoters on the HBV genome (Rossner, 1992, J. Med. Virol. 36, 101-117). We compared their transactivation efficiencies and investigated whether the spatial organization of the promoters with respect to other cis-acting elements might influence their activities. Eight reporter plasmid constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene were designed such that four had the isolated HBV promoters linked to the CAT gene. In the other four, the CAT gene was inserted downstream to each of the four promoters retained in context in the HBV genome. Cells of the human hepatoblastoma line HepG2 were transfected with each one of these reporters together with an effector plasmid, pRSVX, which allowed expression of X protein. All of these promoters could be stimulated by X protein by approximately 2- to 3.5-fold irrespective of their spatial context in the HBV genome. Mutational analysis of in-frame ATG codons in the X gene provides evidence that transactivator product(s) are produced by internal initiation of translation. Transfection of HepG2 cells with HBV genomes bearing a stop mutation in the X gene at codon 118 resulted in poor production of all viral components. Their syntheses were restored upon transfection of the wild-type X gene.

A complementary DNA for human choline acetyltransferase induces two forms of enzyme with different molecular weights in cultured cells

Complementary DNA (cDNA) clones containing the entire coding region of human choline acetyltransferase (ChAT) were isolated from cDNA libraries prepared from the autopsied spinal cord. In the human cDNA, the ATG codon assigned to the putative initiation codon for pig, rat and mouse ChAT cDNAs was replaced by ACG. The human cDNA contained an in-frame ATG codon 324 nucleotides upstream of the ACG codon. Therefore, human ChAT cDNA should code for a 748 amino acid polypeptide of 82.6 kDa. This deduced molecular weight was larger than that of ChAT protein purified from the human brain and placenta (64–70 kDa). The human ChAT cDNA containing the entire coding region was ligated to an expression vector and introduced into African green monkey kidney (COS) cells and Chinese hamster ovary (CHO) cells. The cells expressed high ChAT activity and produced two protein bands immunostained with an antibody to monkey ChAT. The molecular weight of the proteins was estimated to be approximately 70 and 80 kDa by polyacrylamide-SDS gel electrophoresis. When partial cDNAs that lacked the first ATG but contained the replaced ACG codon were introduced into COS cells, the cells expressed moderate ChAT activity and an immunoreactive protein band of 70 kDa. These results indicate that translation of human ChAT mRNA starts at two sites and produces two enzyme proteins with different molecular weights. It might be that the larger form of ChAT molecule is an enzyme precursor for processing or that the N-terminal extrapeptide is needed for subcellular localization of the enzyme.

Molecular cloning, sequence analyses, and expression of complementary DNA encoding murine progesterone receptor

Progesterone receptors exist in two molecular forms commonly designated as "A" and "B" forms, the relative proportion of which can vary among species. In murine tissues, progesterone receptor exists predominantly as the "A" form which, in mammary glands, is also under developmental regulation [Shyamala et al. (1990) Endocrinology 126, 2882-2889]. Therefore, toward resolving the molecular mechanisms responsible for the predominance of the "A" form of progesterone receptor in murine tissues and its developmental regulation, we have isolated, sequenced, and expressed the complementary DNA corresponding to the mouse progesterone receptor. Nucleotide sequence analysis revealed two in-frame ATG codons, such that the largest open reading frame beginning with the first codon could encode a polypeptide with an estimated molecular weight of 99,089, while the shorter open reading frame beginning with the second codon could produce a polypeptide with a calculated molecular weight of 81,829. The murine progesterone receptor had complete identity for the DNA binding domain of human and rabbit progesterone receptors and 99% homology with the chicken progesterone receptor; for the steroid binding domain, it had 96% homology with human and rabbit progesterone receptors and 86% homology with chicken progesterone receptors. Expression of the complete complementary DNA in Chinese hamster ovary cells yielded a protein which bound the synthetic progestin promegestone with an equilibrium dissociation constant of approximately 1 nM, and in Western blot analyses revealed both "A" and "B" forms of immunoreactive receptor.

Bovine beta 1----4-galactosyltransferase: two sets of mRNA transcripts encode two forms of the protein with different amino-terminal domains. In vitro translation experiments demonstrate that both the short and the long forms of the enzyme are type II membrane-bound glycoproteins.

We have used S1 and primer extension analysis to demonstrate that the gene for bovine beta 1----4-galactosyltransferase specifies two sets of mRNA transcripts of different lengths. The longer mRNA transcripts initiate upstream of two in-frame ATG codons and encode a protein of 402 amino acids (long form). The shorter mRNA transcripts initiate between the two in-frame ATG codons and encode a protein of 389 amino acids (short form). These two related forms of beta 1----4-galactosyltransferase have an identical large (358 amino acids), potentially glycosylated, COOH-terminal catalytic domain, and an identical single transmembrane domain. The only difference in primary structure between the two forms is that the long form contains an NH2-terminal extension of 13 amino acids. Thus, bovine beta 1----4-galactosyltransferase fits the pattern established for murine beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. L., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428). Inspection of the NH2-terminal domain suggests that the long form of the bovine enzyme, like its murine counterpart, has a functional cleavable signal sequence which would dictate that the two forms of the membrane-bound enzyme are oriented in opposite directions. We have tested this hypothesis by in vitro translation in the absence or presence of dog pancreas microsomes. In vitro translation of RNA transcripts for the long and short form of beta 1----4-galactosyltransferase in the absence of microsomes results in the synthesis of polypeptides with apparent Mr of 44,500 and 43,000, respectively. In vitro translation of each transcript in the presence of microsomes results in the synthesis of two glycosylated, endoglycosidase H-sensitive proteins with apparent Mr of 47,500 and 46,000. These experiments and additional protease protection experiments demonstrate that the COOH-terminal domain of both the short and the long form of bovine beta 1----4-galactosyltransferase are translocated into the microsomal lumen. By extrapolation, both forms of the enzyme are oriented in vivo as Type II membrane-bound glycoproteins.

Cloning and Sequence Analysis of a Rat Liver cDNA Encoding Acyl-peptide Hydrolase

Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.

Vaccinia virus encodes a thymidylate kinase gene: sequence and transcriptional mapping

The nucleotide sequence and deduced amino acid sequence of a vaccinia virus gene from the SalI F fragment are shown. The predicted polypeptide shares 42% amino acid identity over a 200 amino acid region with Saccharomyces cerevisiae thymidylate kinase (TmpK) and has low homology with herpes simplex virus deoxypyrimidine kinase. Northern blotting and S1 nuclease protection showed that the TmpK gene is transcribed early during infection and mapped the mRNA 5' end to immediately upstream of the second inframe ATG codon of the open reading frame (ORF). The encoded polypeptide is predicted to be 204 amino acids long (23.2 kD) and is almost colinear with yeast TmpK. Vaccinia virus possesses genes for TK and TmpK, separated by 57 kilobases of DNA, which are co-ordinately expressed and the encoded enzymes perform sequential steps in the same biochemical pathway.

Expression and functional analysis of the Rhizobium meliloti nif A gene

The translational initiation point for Rhizobium meliloti nifA, the specific activator for nitrogen fixation (nif) genes, was determined. When expressed in an Escherichia coli linked transcriptional-translational system the DNA coding for the R.meliloti nifA gene produced four polypeptide bands of 71 000, 67 000, 62 000 and 59 000 daltons. There are three in-frame ATG codons at the N-terminus of the gene; by replacing the poor ribosome binding sites of the native DNA with a synthetic consensus ribosome binding site prior to each of ATG codons the polypeptides were produced at enhanced levels and related to each of the initiation codons. The ability of these specifically expressed polypeptides to activate nif promoters fused to lacZ was determined. Only the fulllength polypeptide activated the Klebsiella pneumoniae nifH, R.meliloti nifH and fixA and Bradyrhizobium japonicum nifH and nifD promoters. The R.meliloti fixA promoter, contrary to previous evidence, could be activated in E.coli. Deletion of the putative N-terminal domain of the R.meliloti nifA gene product greatly increased the ability of the protein to activate nif promoters. However, deletions retaining part of this domain were not functional. This shows that the N-terminal domain is not essential for activity and that its presence decreases the full potential function of the protein. Our results are consistent with the suggesting that this domain has a regulatory role. In contrast to K.pneumoniae nifA protein, the function of the full length and domain deleted forms of R.meliloti nifA gene product was sensitive to oxygen in E.coli.

In vivo phosphorylation and protein analysis of hepatitis B virus core antigen.

The C open reading frame of the hepatitis B virus contains two in-frame ATG codons that are separated by the precore region and encodes two major polypeptides that are antigenically distinct and that are probably synthesized from individual mRNAs. The precore region directs the secretion of the e antigen, whereas the core antigen can be expressed in the absence of these sequences. In this report a transient expression system was used to study the hepatitis B virus core antigen. By using a chimeric complex of adenovirus major late promoter-simian virus 40 enhancer sequences, we were able to achieve high levels of core antigen expression in transfected cells, permitting characterization of this protein and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core polypeptide is a 20.9-kilodalton protein, and we show in this study that it is phosphorylated in vivo. Cell fractionation studies, the results of which are supported by indirect immunofluorescence, localized the phosphocore in the cytosol and the nucleus and indicated that it is associated with the membrane of transfected cells. Results of Triton X-114 solubilization studies indicated that the phosphocore is peripherally associated with cytoplasmic membranes. Expression of the membrane-associated phosphocore occurred in the absence of the precore sequences. The phosphocore also assembled into particles in the absence of other viral gene products or intact DNA.

Inducible expression of REP1 causes inducible expression of the 2 micron circle stability system.

The yeast plasmid, 2 micron circle, encodes a stability system consisting of the plasmid replication origin, a cis-active locus designated REP3 and two trans-active functions--the products of the REP1 and REP2 genes. We have constructed 2 micron circle derivatives in which the expression of the REP1 gene is placed under the control of the yeast GAL10 promoter. We show that in such plasmids the stability-system is inducible, being turned off by glucose and turned on by galactose. Further, our results unequivocally demonstrate that, of the two potential in-frame ATG codons at which REP1 translation might initiate (as inferred from the 2 micron circle DNA sequence and from the cap site of the major REP1 transcript), the upstream ATG is dispensable without affecting REP1 function. We also illustrate here a simple and general method for constructing in vivo in yeast 2 micron circle analogs which contain desired alterations within specific regions of the 2 micron circle genome.

Isolation of a cDNA clone encoding the leader peptide of prion protein and expression of the homologous gene in various tissues.

We have isolated a hamster cDNA clone representing the coding sequences for the entire precursor of prion protein (PrP) 27-30. This clone encodes a protein of 254 residues and contains an in-frame ATG codon 42 bases upstream from the one previously reported. Analysis of the predicted amino acid sequence suggests that the PrP precursor protein contains an amino-terminal signal sequence, and a membrane-spanning domain in the carboxyl terminus. Cleavage of the signal peptide would produce a mature protein of 232 amino acids. Sequences homologous to the hamster PrP cDNA were detected in hamster, mouse, sheep, human, and rabbit genomes. A related 2.5-kilobase transcript was present in the brain of normal and scrapie-infected rodents. Two homologous transcripts, 2.5 and 1.1 kilobases, were detected in the lung and heart of normal animals. No PrP mRNA was detected in spleen stroma, a tissue known to contain high titers of scrapie. Antisera raised to the 27- to 30-kDa polypeptide detected the PrP in both normal and infected brains but failed to detect this protein in either normal or infected spleens. Homologous mRNA species were detected in human, sheep, and rabbit brain, even though the latter is resistant to scrapie infection. Our data suggest that PrP is not a necessary component of the infectious agent.

Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.