Conversion of a Golgi apparatus sialyltransferase to a secretory protein by replacement of the NH2-terminal signal anchor with a signal peptide

The beta-galactoside alpha 2,6 sialyltransferase, an integral membrane protein localized to the trans-region of the Golgi apparatus, has been converted into a catalytically active secreted protein by the replacement of the NH2-terminal signal-anchor domain with the cleavable signal peptide of human gamma-interferon. Pulse-chase analysis of the wild type and recombinant proteins expressed in stably transfected Chinese hamster ovary cells showed that the wild type sialyltransferase (47 kDa) remained cell-associated. In contrast, the signal peptide-sialyltransferase fusion protein yielded an enzymatically active 41-kDa polypeptide which was secreted with a half-time of 2-3 h, consistent with cleavage of the signal peptide. The data indicate that the catalytic domain does not contain sufficient information for retention in the Golgi apparatus and that retention signals are likely to be found in the NH2-terminal 57 amino acids of the wild type enzyme.     

Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme

Rat liver alpha-mannosidase II, a hydrolase involved in the processing of asparagine-linked oligosaccharides, is an integral membrane glycoprotein facing the lumen of Golgi membranes. We have previously shown (Moremen, K. W., and Touster, O. (1986) J. Biol. Chem. 261, 10945-10951) that mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes will result in the cleavage of the intact 124,000-dalton alpha-mannosidase II subunit, releasing a 110,000-dalton hydrophilic polypeptide which contains the catalytic site. Consistent with the removal of a membrane binding domain, the chymotrypsin-generated 110,000-dalton peptide was found exclusively in the aqueous phase in Triton X-114 phase separation studies, whereas the intact enzyme was found in the detergent phase. Taking advantage of this conversion in phase partitioning behavior, a purification procedure was developed to isolate the 110,000-dalton proteolytic digestion product as a homogeneous polypeptide for further characterization and protein sequencing at a yield of greater than 65% from a rat liver Golgi-enriched membrane fraction. An improved purification procedure for the intact enzyme was also developed. The two forms of the enzyme were compared yielding the following results. (a) The catalytic activity of the intact and cleaved forms of alpha-mannosidase II were indistinguishable in Km, Vmax, inhibition by the alkaloid, swainsonine, and in their activity toward the natural substrate GlcNAc-Man5GlcNAc. (b) Both the intact and cleaved forms of the enzyme appear to be disulfide-linked dimers. (c) The two forms of the enzyme contain different NH2-terminal sequences suggesting that the cleaved NH2 terminus contains the membrane-spanning domain. (d) Additional peptide sequences were obtained from proteolytic fragments and cyanogen bromide digestion products in order to create a partial protein sequence map of the enzyme. These results are consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored to the lumenal face of Golgi membranes through an NH2-terminal hydrophobic membrane-anchoring domain.     

Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae.

A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced. The DNA sequence encodes a precursor protein containing 387 amino acid residues. The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids. The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established. In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases. Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.     

Purification and complete sequence of a small proteolipid associated with the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The purified plasma membrane H(+)-ATPase of Schizosaccharomyces pombe and Saccharomyces cerevisiae display, in addition to the catalytic subunit of 100 kDa, a highly mobile component, soluble in chloroform/methanol. Chloroform/methanol extraction of S. cerevisiae plasma membranes led to isolation of a low molecular weight proteolipid identical to that present in purified H(+)-ATPase. NH2-terminal amino acid sequencing revealed a 38-residue polypeptide with a calculated molecular mass of 4250 Da. The polypeptide lacks the first two NH2-terminal amino acids as compared with the deduced sequence of the PMP1 gene (for plasma membrane proteolipid) isolated by hybridization with an oligonucleotide probe corresponding to an internal amino acid sequence of the proteolipid. The polypeptide is predicted to contain an NH2-terminal transmembrane segment followed by a very basic hydrophilic domain.     

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N-glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.     

A yeast Golgi E-type ATPase with an unusual membrane topology.

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.     

Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA

LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes.     

The 17-residue transmembrane domain of beta-galactoside alpha 2,6- sialyltransferase is sufficient for Golgi retention

beta-Galactoside alpha 2,6-sialyltransferase (ST) is a type II integral membrane protein of the Golgi apparatus involved in the sialylation of N-linked glycans. A series of experiments has shown that the 17-residue transmembrane domain of ST is sufficient to confer localization to the Golgi apparatus when transferred to the corresponding region of a cell surface type II integral membrane protein. Lectin affinity chromatography of chimeric proteins bearing this 17-residue sequence suggests that these chimeric proteins are localized in the trans-Golgi cisternae and/or trans-Golgi network. Further experiments suggest that this 17-residue sequence functions as a retention signal for the Golgi apparatus.     

Cloning and expression of the Gal beta 1, 3GalNAc alpha 2,3-sialyltransferase.

Sequence information obtained by NH2-terminal sequence analysis of two molecular weight forms (45 and 48 kDa) of the porcine Gal beta 1,3GalNAc alpha 2,3-sialyltransferase was used to clone a full-length cDNA of the enzyme. The cDNA sequence revealed an open reading frame coding for 343 amino acids and a putative domain structure consisting of a short NH2-terminal cytoplasmic domain, a signal-anchor sequence, and a large COOH-terminal catalytic domain. This domain structure was confirmed by construction of a recombinant sialyltransferase in which the cytoplasmic domain and signal-anchor sequence of the enzyme was replaced with the cDNA of insulin signal sequence. Expression of the resulting construct in COS-1 cells produced an active sialyltransferase which was secreted into the medium in soluble form. Comparison of the cDNA sequence of the sialyltransferase with GenBank produced no significant homologies except with the previously described Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase. Although the cDNA sequences of these two enzymes were largely nonhomologous, there was a 45-amino acid sequence which exhibited 65% identity. This observation suggests that the two sialyltransferases were derived, in part, from a common gene.     

Primary structure of rat liver dipeptidyl peptidase IV deduced from its cDNA and identification of the NH2-terminal signal sequence as the membrane-anchoring domain

Two forms of dipeptidyl peptidase IV (DPP) were purified from rat liver plasma membranes: a membrane form (mDPP) extracted with Triton X-100 and a soluble form (sDPP) prepared by treatment with papain. Apparent molecular masses of mDPP and sDPP were 109 and 105 kDa, respectively, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal sequences of the two forms were found to be completely different from each other. For further information on the molecular structure, we constructed a lambda gt11 liver cDNA library and isolated two cDNA clones for DPP, lambda cDP37 and lambda cD5. The 3.5-kilobase cDNA insert of lambda cDP37 contains an open reading frame that encodes a 767-residue polypeptide with a calculated size of 88,107 Da, which is in reasonable agreement with that of DPP (87 kDa) immunoprecipitated from cell-free translation products. Eight potential N-linked glycosylation sites were found in the molecule, accounting for the difference in mass between the precursor and mature forms. Of particular interest is that the deduced NH2-terminal sequence with a characteristic signal peptide is completely identical to that determined for mDPP. In addition, the NH2-terminal sequence of sDPP is identified in the predicted sequence starting at the 35th position from the NH2 terminus. These results indicate that the signal peptide of DPP is not cleaved off during biosynthesis but functions as the membrane-anchoring domain even in the mature form. It is also found that the primary structure thus predicted has striking homology to that of gp 110, a bile canaliculus domain-specific membrane glycoprotein (Hong, W., and Doyle, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7962-7966).     

Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum

We present and evaluate a model for the secondary structure and membrane orientation of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the glycoprotein of the endoplasmic reticulum that controls the rate of cholesterol biosynthesis. This model is derived from proteolysis experiments that separate the 97-kilodalton enzyme into two domains, an NH2-terminal membrane-bound domain of 339 residues and a COOH-terminal water-soluble domain of 548 residues that projects into the cytoplasm and contains the catalytic site. These domains were identified by reaction with antibodies against synthetic peptides corresponding to specific regions in the molecule. Computer modeling of the reductase structure, based on the amino acid sequence as determined by molecular cloning, predicts that the NH2-terminal domain contains 7 membrane-spanning regions. Analysis of the gene structure reveals that each proposed membrane-spanning region is encoded in a separate exon and is separated from the adjacent membrane-spanning region by an intron. The COOH-terminal domain of the reductase is predicted to contain two beta-structures flanked by a series of amphipathic helices, which together may constitute the active site. The NH2-terminal membrane-bound domain of the reductase bears some resemblance to rhodopsin, the photoreceptor protein of retinal rod disks and the only other intracellular glycoprotein whose amino acid sequence is known.     

The signal anchor and stem regions of the beta-galactoside alpha 2,6-sialyltransferase may each act to localize the enzyme to the Golgi apparatus.

The beta-galactoside alpha 2,6-sialyltransferase has been localized to the trans cisternae of the Golgi apparatus and the trans Golgi network where it transfers sialic acid residues to terminal positions on N-linked oligosaccharides. It is a type II transmembrane protein possessing a 9-amino acid amino-terminal cytoplasmic tail, a 17-amino acid signal anchor domain, and a 35-amino acid stem region which tethers the large luminal catalytic domain to the membrane anchor. Previous work has demonstrated that the soluble sialytransferase catalytic domain is rapidly secreted from Chinese hamster ovary cells. These results suggest that the signals for Golgi apparatus localization do not reside in the catalytic domain of the enzyme but must reside in the cytoplasmic tail, signal anchor domain, and/or stem region. To determine which amino-terminal regions are required for Golgi apparatus localization, mutant sialyltransferase proteins were constructed by in vitro oligonucleotide-directed mutagenesis, expressed in Cos-1 cells, and localized by indirect immunofluorescence microscopy. Signal cleavage-sialyltransferase mutants which consist of only the stem and catalytic domain of the enzyme are not rapidly secreted but are retained intracellularly and predominantly localized to the Golgi apparatus. However, deletion of either the stem region or the cytoplasmic tail of the membrane-bound sialyltransferase does not alter its Golgi apparatus localization. In addition, sequential replacement of the amino acids of the sialyltransferase signal anchor domain with amino acids from the signal anchor domain of a plasma membrane protein, the influenza virus neuraminidase does not alter the Golgi apparatus localization of the sialyltransferase. These observations suggest that sequences in the signal anchor region and stem region allow the Golgi apparatus localization of the membrane-bound and soluble forms of the sialytransferase, respectively, and that both regions may contain Golgi apparatus localization signals.     

Primary structure of human placental 5'-nucleotidase and identification of the glycolipid anchor in the mature form

A cDNA was cloned coding for human placental 5'-nucleotidase. The 3547-bp cDNA contains an open reading frame that encodes a 574-residue polypeptide with calculated size of 63 375 Da. The NH2-terminal 26 residues comprise a signal peptide, which is followed by the NH2-terminal sequence of the purified protein. four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5'-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-Gly-Arg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510-523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5'-nucleotidase lacks the predicted COOH-terminal peptide extension (524-548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5'-nucleotidase.     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

The Sec14 homology domain regulates the cellular distribution and transforming activity of the Rho-specific guanine nucleotide exchange factor Dbs

Dbs is a Rho-specific guanine nucleotide exchange factor that was identified in a screen for proteins whose overexpression cause deregulated growth in murine fibroblasts. Dbs contains multiple recognizable motifs including a centrally located Rho-specific guanine nucleotide exchange factor domain, a COOH-terminal Src homology 3 domain, two spectrin-like repeats, and a recently identified NH(2)-terminal Sec14 homology domain. The transforming potential of Dbs is substantially activated by the removal of inhibitory sequences that lie outside of the core catalytic sequences, and in this current study we mapped this inhibition to the Sec14 domain. Surprisingly removal of the NH(2) terminus did not alter the catalytic activity of Dbs in vivo but rather altered its subcellular distribution. Whereas full-length Dbs was distributed primarily in a perinuclear structure that coincides with a marker for the Golgi apparatus, removal of the Sec14 domain was associated with translocation of Dbs to the cell periphery where it accumulated within membrane ruffles and lamellipodia. However, translocation of Dbs and the concomitant changes in the actin cytoskeleton were not sufficient to fully activate Dbs transformation. The Sec14 domain also forms intramolecular contacts with the pleckstrin homology domain, and these contacts must also be relieved to achieve full transforming activity. Collectively these observations suggest that the Sec14 domain regulates Dbs transformation through at least two distinct mechanisms, neither of which appears to directly influence the in vivo exchange activity of the protein.     

Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation.

We have deduced the entire 575-amino acid sequence of the human thrombomodulin precursor from cDNA clones. The precursor starts with an 18-residue signal peptide domain, followed by the NH2-terminal domain, a domain with six epidermal growth factor-like structures, an O-glycosylation site-rich domain, a 24-residue transmembrane domain and a cytoplasmic domain. Simian COS cells transfected with the expression vector pSV2 containing thrombomodulin cDNA synthesized immunoreactive and functionally active thrombomodulin.