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Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum.

ABSTRACT: A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10). The inserts of the immunoreactive clones were short (0.9-1.1 kbp). One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx. 1.9 kbp) and widely overlapping cDNA inserts. They did not contain the first two nucleotide residues of the initiator codon, nor the 5'-end untranslated portion of the mRNA. These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5'-end of the already known cDNA sequence. The nucleotide sequence consists of 48 bp of 5'-end non-coding region, 1695 bp of coding region and 212 bp of 3'-end non-coding region including a 20 bp poly(A) tail. The signal peptide and the mature protein subunit are 18 and 547 residues long respectively. Tyr is confirmed as N-terminal residue. The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing [Korza & Ozols (1988) J. Biol. Chem. 263, 3486-3495; Ozols (1989) J. Biol. Chem. 264, 12533-12545]. The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus. The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells [Munro & Pelham (1987) Cell 48, 899-907]; -HDEL in yeast [Pelham, Hardwick & Lewis (1988) EMBO J. 7, 1757-1762]). We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum. In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.

SUBMITTER: Robbi M

PROVIDER: S-EPMC1131598 | biostudies-other | 1990 Jul

REPOSITORIES: biostudies-other

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Project description:The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues -1 and -3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic alpha-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37(M), a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH(2)-terminal fragment, pUL37(M-NH2), was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37(M-COOH), was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH(2)-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37(M), and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.

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Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum.

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