(Received for publication, December 7, 1995)
From the
Mannan chains of Kluyveromyces lactis mannoproteins are
similar to those of Saccharomyces cerevisiae except that they
have terminal 1
2-linked N-acetylglucosamine and
lack mannose phosphate. In a previous study, Douglas and Ballou
(Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21,
1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had
normal levels of N-acetylglucosaminyltransferase activity, and
the partially purified enzyme from wild-type and mutant cells had the
same apparent size, heat stability, affinity for substrates, metal
requirement, and subcellular location. No qualitative or quantitative
differences were found between mutant and wild-type cells in endogenous
mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at
similar rates in wild-type and mutant cells, and the latter did not
have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could
remove N-acetylglucosamine from mannoproteins. Together, the
above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a
defect in compartmentation of substrates involved in the biosynthesis
of mannoproteins.
We determined whether the above mutant phenotype
is the result of defective transport of UDP-GlcNAc into Golgi vesicles
from K. lactis. Golgi vesicles which were sealed and of the
same membrane topographical orientation as in vivo were
isolated from wild-type and mnn2-2 mutant cells and incubated with
UDP-GlcNAc in an assay in vitro. The initial rate of transport
of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature
dependent, saturable with an apparent K of 5.5 µM and a V
of 8.2
pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into
Golgi vesicles from mutant cells. However, Golgi vesicles from both
cells translocated GDP-mannose at comparable velocities, indicating
that the above transport defect is specific. In addition to the above
defect in mannoproteins, mutant cells were also deficient in the
biosynthesis of glucosamine containing lipids.
Mannan chains of Kluyveromyces lactis mannoproteins are
similar to those of Saccharomyces cerevisiae except that they
have terminal 1
2-linked N-acetylglucosamine units
and lack mannose phosphate groups(1) . Ballou and co-workers
isolated and characterized mutants of K. lactis without
terminal N-acetylglucosamine in their
mannoproteins(2) . One mutant, mnn2-1, lacks the terminal N-acetylglucosaminyltransferase activity while the other
mutant, mnn2-2, has wild-type levels of this enzyme. This latter
observation prompted Douglas and Ballou (3) to study the
biochemical defect underlying this latter phenotype. They found that
wild-type and mutant cells had similar pool sizes of UDP-GlcNAc,
synthesized chitin at similar rates, and that endogenous mannan
acceptors were the same in both cell types. Highly purified
preparations of N-acetylglucosaminyltransferase were obtained
from both cells and were found to have the same apparent size, heat
stability, apparent K
values for
substrates, Mn
requirements, V
, and subcellular location. No evidence was
found for an inactive enzyme precursor in mutant cells nor did these
cells contain a soluble inhibitor of the transferase or a
hexosaminidase which could remove N-acetylglucosamine from the
mannoproteins during their biosynthesis and secretion. These
observations led the above authors to postulate, among other
possibilities, that the mutant might have a defect in compartmentation
of substrates thereby preventing the normal biosynthesis of the
mannoproteins.
We determined whether the above mutant phenotype of K. lactis is the result of defective transport of UDP-GlcNAc into Golgi vesicles by comparing the initial rates of transport of UDP-GlcNAc into Golgi vesicles from wild-type and mutant mnn2-2 and mnn2-1 cells. Vesicles from wild-type and mutant mnn2-1 cells were able to transport in vitro UDP-GlcNAc in a temperature-dependent and saturable manner while no transport of UDP-GlcNAc into vesicles from mutant mnn2-2 cells was detected. However, vesicles from all these cells translocated GDP-mannose at a comparable velocity; this indicates that the above defect for UDP-GlcNAc transport is specific. In addition to their previously reported lack of N-acetylglucosamine in outer chain of mannoproteins, mnn2-2 mutant cells were also found deficient in their biosynthesis of glucosamine containing lipids. This phenotype is not secondary to the lack of GlcNAc in glycoproteins since mnn2-1, the glycoprotein GlcNAc-transferase mutant, has the same GlcNAc containing lipids as wild-type.
Yeast strains were grown at 30 °C in shaker liquid culture consisting of 1% yeast extract, 2% bactopeptone, containing 2% glucose (YPD) unless otherwise specified. Cultures were maintained on solid YPD media containing 2% Bacto-agar.
Lipids were analyzed by ascending thin
layer chromatography on 0.2-mm Silica Gel 60 plates (EM Science) in a
solvent system of chloroform, methanol, 0.2% aqueous KCl (55:45:10).
Approximately 40,000 cpm were applied per sample. The developed plates
were sprayed with ENHANCE (DuPont NEN) and fluorograms were
exposed on Reflection
film (DuPont NEN) for 3 weeks at
-80 °C. To determine sensitivity to mild alkaline hydrolysis,
an aliquot of the lipid extract was dried under nitrogen and
resuspended in ethanol/water/diethyl ether/pyridine (15:15:5:1)
(v/v/v/v). 100 µl of 0.2 N KOH in methanol was added.
Following 1 h incubation at room temperature, 20 µl of 1 N acetic acid was added to neutralize the reaction. The mixture was
evaporated under nitrogen, desalted, and separated on thin layer
chromatography as described above.
As can be seen in Table 2, while transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was concentration dependent, transport into Golgi vesicles from mnn2-2 mutants was virtually zero and did not change with a 10-fold increase in nucleotide sugar concentration. Vesicles from the mnn2-1 mutant translocate UDP-GlcNAc at rates comparable to vesicles from wild-type cells.
It was important to determine that the above defect in
UDP-GlcNAc transport was specific and not a general defect of the Golgi
membrane of mutant mnn2-2 cells. We therefore measured the ability of
vesicles to transport GDP-mannose. As can be seen in Table 2,
this nucleotide sugar was translocated into all vesicles with a similar
velocity, demonstrating that the above defect of UDP-GlcNAc transport
was specific. The V of GDP-mannose transport
into K. lactis vesicles was similar to that previously found
with vesicles from S. cerevisiae; this was expected as both
yeasts have similar mannan chains.
Figure 1:
Rate of solute
accumulation within wild-type K. lactis Golgi vesicles versus UDP-GlcNAc concentration in the incubation medium. A
P vesicle fraction (1.2 mg of protein) was incubated at 30
°C for 3 min with different concentrations of
UDP-[
H]GlcNAc (600 dpm/pmol) in a final volume of
1 ml. Results are the mean of two separate
determinations.
Figure 2:
[H]Glucosamine-containing
lipids of wild-type and mutant mnn2-2 K. lactis. Wild-type and
mutant cells were grown in the presence of
[
H]glucosamine as described under
``Materials and Methods.'' Total lipids (lanes 1 and 4) and lipids following mild alkaline hydrolysis (lanes 2 and 3) were separated by thin layer chromatography in
chloroform, methanol, 0.22% aqueous KCl (55:45:10) and visualized by
fluorography after 27 days.
Figure 3:
[H]Glucosamine-containing
lipids of wild-type and mutant mnn2-1 K. lactis. Cells were
grown in the presence of [
H]glucosamine as
described under ``Materials and Methods.'' Total lipids (lanes 1 and 4) and those following mild alkaline
hydrolysis (lanes 2 and 3) were separated as
described in the legend of Fig. 2. Visualization by fluorography
was after 10 days.
This study demonstrates that the N-acetylglucosamine-deficient phenotype of the mnn2-2 mutant
of K. lactis is the result of impaired transport of UDP-GlcNAc
into Golgi vesicles. This is the first mutant yeast cell line
characterized as deficient in Golgi transport of any solute and differs
from known nucleotide sugar transport mutants of mammalian cells since
the latter show 5% of Golgi in vitro transport activity
compared to their corresponding wild-type vesicles and also have
greatly reduced, but not zero, levels of the corresponding sugar in
proteins, proteoglycans, and glycolipids. Because both wild-type and
mnn2-2-derived Golgi vesicles from K. lactis transport
GDP-mannose at comparable rates, this study strongly suggests that the
defect is specific. The apparent K for UDP-GlcNAc
transport into Golgi vesicles from wild-type K. lactis is
comparable to that of other nucleotide sugars such as GDP-mannose into
Golgi vesicles from S. cerevisiae(9) . Although S.
cerevisiae does not have N-acetylglucosamine in its outer
chains and therefore would not be expected to require a Golgi
UDP-GlcNAc transporter for glycoproteins, mammalian cells have been
shown in previous studies in vitro to possess such transport
activity both in the Golgi apparatus (17) and the endoplasmic
reticulum (17, 18) . The latter organelle presumably
uses UDP-GlcNAc for the biosynthesis O-GlcNAc containing
glycoproteins. Other yeast strains, such as Schizosaccharomyces
pombe, contain galactose in their outer chains which presumably is
transported into the Golgi lumen via UDP-galactose.
Although biochemical and genetic studies with mammalian cells strongly suggest that the different uridine containing nucleotide sugars are transported into the lumen of the Golgi via individual specific transporters(12, 13, 14) , it will be important to determine whether there are structural similarities between the UDP-GlcNAc transporter of K. lactis, the presumed UDP-galactose transporter of S. pombe, mammalian UDP-GlcNAc transporters of the Golgi and endoplasmic reticulum membrane, as well as other uridine sugar nucleotide transporters (see below). Mammalian studies suggest that UMP is the antiporter for UDP-GlcNAc and UDP-galactose and that this nucleoside monophosphate is a competitive inhibitor of both transporters suggesting that they both may contain common structural features(19, 20) .
The antiporter for yeast Golgi transport of GDP-mannose has been characterized as being GMP; this molecule arises by the action of a Golgi lumenal GDPase. Biochemical, cell biological, and genetic studies have shown that the GDPase plays a pivotal role in the entry of GDP-mannose into the Golgi and subsequent mannosylation of proteins and lipids in vivo(10, 16, 21) . Previous studies in vitro(4) have also shown that this GDPase has significant UDPase activity; thus, it is possible that the same GDPase protein may give rise to UMP, the putative antiporter for the UDP-GlcNAc transporter in K. lactis. At this time, it is not clear whether this yeast has a UDPase protein separate from the GDPase or whether the latter protein also mediates the in vivo degradation of UDP to UMP.
The availability of the yeast mutant described here makes this strain an attractive candidate for the cloning of the corresponding transporter. This in turn should allow determination of whether the transporter of UDP-GlcNAc for K. lactis is homologous to other yeast and mammalian transporters.