HUMAN BETA-1,4 GALACTOSYLTRANSFERASE AND ALPHA-2,6 SIALYLTRANSFERASE EXPRESSED IN SACCHAROMYCES-CEREVISIAE ARE RETAINED AS ACTIVE ENZYMES IN THE ENDOPLASMIC-RETICULUM

Biosynthesis and intracellular transport of recombinant human full-len gth beta 1,4 galactosyltransferase (GT) and full-length alpha 2,6 sial yltransferase (ST) were investigated in Saccharomyces cerevisiae. Rece ntly, enzymic activity of recombinant GT (rGT) in crude homogenates of S. cerevisiae could successfully be demonstrated [Krezdorn, C., Watze le, G., Kleene, R. B., Ivanov, S. X. and Berger, E. G. (1993) Eur J. B iochem. 212, 113-120]. In the present work, we show that, in yeast str ains transformed with plasmid pDPSIA containing the cDNA coding for hu man ST, rST enzymic activity using asialo-fetuin or N-acetyllactosamin e as acceptor substrates could readily be detected. Analysis by H-1-NM R spectroscopy of the disaccharide product of rGT as recently reported , and the trisaccharide product of rST demonstrated that only the expe cted glycosidic linkages were formed. Following mechanical disruption of yeast cells, both enzymes sedimented with a fraction enriched in me mbranes of the endoplasmic reticulum (ER) and were activated by Triton X-100 3-5-fold. rGT and rST could be immunoprecipitated from their [S -35]Met-labelled transformed yeast extracts using polyclonal antibodie s raised against fusion proteins consisting of beta-galactosidase-GT o r beta-galactosidase-ST, respectively, expressed in Escherichia coli. For rGT a single glycosylated form of apparent molecular mass 48 kDa w as reported, but for rST two main bands corresponding to apparent mole cular mas ses of 48 kDa and 44 kDa, respectively, were detected. Immun oprecipitation from either tunicamycin-treated [S-35]Met-labelled tran sformed yeast cells or labelling with radioactive sugars both indicate d that the 44-kDa form of rST was non-glycosylated and that the 48-kDa form of rST was core N-glycosylated. In addition, core glycosylation of both recombinant enzymes demonstrated that they were competent for translocation across the ER membranes. However, the 44-kDa form of rST was converted to the 48-kDa glycosylated form only slowly, suggesting a mechanism of posttranslational translocation. Absence of hyperglyco sylation of rST and rGT in wild type and lack of the Golgi-specific ma n-alpha 1,6-man epitope suggest that the recombinant enzymes did not e nter the yeast Golgi apparatus. These results indicated that both rGT and rST are retained as enzymically active enzymes in the ER of yeast and suggest a ribonucleoprotein-independent import of rST into the ER.