Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

The in vitro transcription/translation of the clone MR-8 cDNA also resulted in a band of identical mobility to the deglycosylated 3T3 enzyme (Fig . 3) . Translation in vitro in the presence of microsomal membranes resulted in the glycosylation of the polypeptide and a decrease in mobility to a doublet of identical size to that of the glycosylated 3T3 . Comparison of murine Man II protein sequence with the yeast vacuolar a-mannosidase and the rat ER a-mannosidase. Murine Man II was aligned with the yeast vacuolar a-mannosidase and the rat ER a-mannosidase using the FASTA program (54) . The sequences in the figure show the individual alignments of murine Man II (center sequence) with the yeast vacuolar a-mannosidase (59) (Yeast Man, upper sequence) and the rat ER a-mannosidase (24) (ER Man, lower sequence) . The vertical bar indicates amino acid identities between the two lines of sequence . The double dots indicate conservative amino acid substitutions between the two lines of sequence . The numbering below the aligned sequences refers to the polypeptide numbering of 3T3 Man II in Fig . 2 . When the indicated sequence pairs were subjected to statistical analysis using the Bestfit program from the University of Wisconsin Genetics Computer Group (comparison versus randomized sequences with the same amino acid content, gap weight = 3, gap length weight = 0.1) the sequence alignments were found to be 9 .7 and 6.0 SDs above random for the Man II/yeast vacuolar a-mannosidase pair and the Man II/ER a-mannosidase, respectively. biosynthetic product . The doublet likely results from the glycosylation of Asn residue 1,129 and a partial glycosylation at Asn residues 78 or 93, since the purified soluble form of the rat liver enzyme (residues 107-1,150) has been shown to be fully glycosylated at a single site (30) . The apparent molecular weight on SDS gels is slightly less than is predicted for the open reading frame of the cDNA (rv117 kD vs 131 kD for the deglycosylated forms, respectively) . This presumably results from an anomalous gel migration since the in vitro translation product and the product of biosynthetic labeling run at an identical position by SDS-PAGE . Translation in the presence of membranes also resulted in the apparent co-translational translocation of the polypeptide across the lipid bilayer. Trypsin cleavage of the in vitro translation products in the presence of microsomal membranes did not alter the amount or the mobility of the glycosylated form but the smaller in vitro-synthesized products were fully susceptible to digestion . The lack of a mobility shift following trypsin digestion also confirms the orientation in the membrane and the location of the membrane-spanning domain . These results are also consistent with the inability to detect any cytoplasmically oriented polypeptide in protease protection experiments in intact Golgi membranes (27) . Addition of membranes posttranslationally did not result in a significant production of glycosylated product suggesting a requirement for co-translational translocation of the enzyme .