HUMAN BETA-1,4 GALACTOSYLTRANSFERASE AND ALPHA-2,6 SIALYLTRANSFERASE EXPRESSED IN SACCHAROMYCES-CEREVISIAE ARE RETAINED AS ACTIVE ENZYMES IN THE ENDOPLASMIC-RETICULUM
Ch. Krezdorn et al., HUMAN BETA-1,4 GALACTOSYLTRANSFERASE AND ALPHA-2,6 SIALYLTRANSFERASE EXPRESSED IN SACCHAROMYCES-CEREVISIAE ARE RETAINED AS ACTIVE ENZYMES IN THE ENDOPLASMIC-RETICULUM, European journal of biochemistry, 220(3), 1994, pp. 809-817
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.