Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, 700 Albany Street Boston, MA 02118, USA
Received on November 15, 2000; revised on January 17, 2001; accepted on January 17, 2001.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: yeast/Golgi/glycosylation/nucleoside diphosphatases
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nucleoside diphosphatase activities have also been described in mammalian membranes of the Golgi apparatus (Brandan and Fleischer, 1982; Wang and Guidotti, 1998
) and endoplasmic reticulum (ER; Trombetta and Helenius, 1999
; Ohkubo et al., 1980
). Two related nucleoside diphosphatases, hypothesized to have a role in generating nucleoside monophosphates in the lumen of the ER and Golgi apparatus, have recently been cloned. An ecto (E)-ATPase from human brain was shown to localize to the Golgi apparatus of transfected COS 7 cells (Wang and Guidotti, 1998
). This protein has high activity toward UDP; lower toward GDP, CDP, and TDP; and lowest toward the corresponding nucleoside triphosphates. Trombetta and Helenius (1999)
recently purified and cloned a rat liver ER nucleoside diphosphatase that hydrolyzes UDP, GDP, and IDP but no other nucleoside phosphates. These mammalian nucleoside diphosphatases share high sequence similarity in four conserved apyrase regions with Gdalp and other proteins of still unidentified function. A Golgi GDPase has also been detected in plants and hypothesized to yield GMP, the putative antiporter for GDP-fucose transport into the Golgi lumen (Wulff et al., 2000
).
We showed previously that the S. cerevisiae Golgi Gda1p could hydrolyze UDP, in addition to GDP, albeit at considerably slower rate. In the presence of Mn2+ ions, the UDPase activity was 33% of GDPase activity, whereas in the presence of Ca2+ ions it was 11% (Yanagisawa et al., 1990). The role of this UDPase activity is not clear because S. cerevisiae solely utilizes GDP-mannose as sugar donor in the Golgi lumen and no reaction utilizing uridine nucleotide sugars has been described in the Golgi apparatus of this organism.
Kluyveromyces lactis is a yeast species closely related to S. cerevisiae; its outer mannan chains differ from those of S. cerevisiae by having terminal N-acetylglucosamine and no mannose phosphate (Raschke and Ballou, 1972). Previous studies from our laboratory characterized a mutant of K. lactis that lacked terminal N-acetylglucosamine as a consequence of a deficiency in the Golgi UDP-N-acetylglucosamine transporter (Abeijon et al., 1996a
). Phenotypic correction of this mutant allowed the molecular cloning of the first nucleotide sugar transporter gene (Abeijon et al., 1996b
) which was later found to be contiguous to the N- acetylglucosaminyttransferase that catalyzes the addition of the terminal N- acetylglucosamine to the K. lactis outer mannan chains (Hirschberg et al., 1998
).
K. lactis uses both guanosine and uridine nucleotide sugars for synthesis of its sugar polymers in the lumen of its Golgi apparatus. We were therefore interested in determining whether it has a nucleoside diphosphatase with both GDPase and UDPase activities in the Golgi lumen, and if so, its role in transport of GDP-mannose and UDP-GlcNAc into Golgi vesicles.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
If KlGda1p is also a UDPase in vivo, one would predict that the null mutant would have a reduction in transport of UDP-GlcNAc into K. lactis Golgi vesicles, in addition to that of GDP-mannose. As can be seen in Figure 3, transport of UDP-GlcNAc into the lumen of Golgi-enriched vesicles from K. lactis Klgda1 null mutants was approximately half compared to that into wild-type vesicles. This reduction was seen in both solutes within vesicles as well as in sugars bound to macromolecules within the vesicles. Transport of GDP-mannose and UDP-glucose was also reduced (Figure 3). Reduction of UDP-glucose transport had not been observed in S. cerevisiae gda1 null mutants (not shown), suggesting that K. lactis may have a UDP-glucose transport activity in both the Golgi apparatus and endoplasmic reticulum. In S. cerevisiae UDP-glucose enters primarily into the endoplasmic reticulum (Castro et al., 1999), whereas Gda1p resides in the Golgi.
|
|
|
We then used chitinase, a heavily O-glycosylated protein that is secreted into the media, as a probe for changes in O-glycosylation. As can be seen in Figure 6 lanes 4 and 5, chitinase from the null mutant migrated faster than that of wild-type cells. We had previously shown that in S. cerevisiae gda1 null mutants this faster migration of chitinase on SDS gels was the result of decreased O-mannosylation (Abeijon et al., 1993). The same behavior was observed here (Figure 6 lanes 1 and 2). We also observed a slight increase of shorter mannose chains on ß-elimination of total glycoproteins from the null mutant compared to wild-type cells (not shown). Finally, we tested the ability of a plasmid containing KlGDA1 to correct the mannosylation defect of chitinase expressed by the S. cerevisiae null mutants. As can be seen in Figure 6, lane 3, S. cerevisiae chitinase regained its wild-type mobility, demonstrating functional homology between the S. cerevisiae and K. lactis GDA1 genes.
|
|
The different lysis behavior of cells following ZymolyaseTM treatment suggested differences in cell wall composition. To test this hypothesis, alkali-insoluble glucans were prepared from cell walls of wild-type and gda1 null mutants of S. cerevisiae and K. lactis. Deletion of GDA1 in K. lactis resulted in an increase of cell wall ß-1,3 glucans, relative to wild type, in cells grown at both 23°C and 37°C. A more robust change was seen at the latter temperature (Figure 8, top). An increase of ß-1,6 glucans was also observed in K. lactis gda1 mutants, but only when grown at 37°C. In S. cerevisiae, contrary to K. lactis, null mutants did not show an increase of ß-1,3 glucans but a decrease; this was more pronounced in cells grown at 23°C than 37°C and was also observed to a lesser extent for ß-1,6 glucans. Chitin, another cell wall component, was not changed in the K. lactis gda1 null mutant but showed a twofold increase in the corresponding mutant of S. cerevisiae (Figure 9).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Deletion of KlGDA1 resulted in some residual GDPase activity in the K. lactis membranes, while almost no remaining activity had been detected in membranes of S. cerevisiae gda1 null mutants (Abeijon et al., 1993). Even though a very low nucleoside diphosphatase activity can be measured in vitro in S. cerevisiae gda1 null mutants, another apyrase, Ynd1p, has recently been identified and cloned in this organism (Guillen et al., 1999
). The substrate specificity of Ynd1p is broader than that of Gda1p, but their function is apparently partially redundant because the double mutant gda1
ynd1
has a more severe glycosylation phenotype than any of the individual mutants (Guillen et al., 1999
). Ynd1p apyrase activity in the Golgi lumen has been shown to have a very narrow optimal pH, between 7.5 and 8, and to be down-regulated by direct binding of its cytosolic domain to the activator subunit Vma13p of the vacuolar H+ ATPase (Zhong et al., 2000
). Most likely, K. lactis has at least one other nucleoside diphosphatase or apyrase that generates nucleoside monophosphates in its Golgi lumen. This could explain why N-glycosylation was not affected in KlGDA1 mutants.
The possibility of multiple proteins in the Golgi lumen giving rise to nucleoside monophosphates is particularly relevant to mammalian Golgi membranes where evidence already suggests that more that one protein with nucleoside diphosphatase activity may reside (Wang and Guidotti, 1998). These observations, in addition to results of this study, strongly suggest that there is regulation of glycosylation also at the level of the production of the antiporter molecules involved in the nucleotide sugar transport cycle in higher and lower eukaryotes.
These studies are the first to show a role for GDA1 in yeast cell wall morphogenesis. Surprisingly, despite of the amino acid sequence similarity between ScGDA1 and KlGDA1 and their functional homology, deletion of each gene had very different effects on osmotic stability and cell wall polymer composition of both null mutant strains compared to each other and to their respective wild types. These differences were even more accentuated when the cells were grown at 37°C instead of 23°C. Thus, K. lactis gda1 null mutants were more sensitive toward ß-1,3 glucanaseinduced cell lysis than their wild type possibly because they had increased cell wall ß-1,3 glucans at both temperatures. ß-1,6 glucans were increased only when mutants were grown at 37°C, and chitin was unchanged at both growth temperatures. In marked contrast, S. cerevisiae deletion mutants were more resistant toward ß-1,3 glucanaseinduced cell lysis than wild type at both temperatures. This behavior may be a consequence of increased cell wall chitin content found in S. cerevisiae mutants grown at both temperatures. Chitin fibers may give strength to the wall and make it more resistant to osmotic shock, even in the presence of a decrease in ß-1,3 glucans. The changes found in the cell wall of S. cerevisiae gda1 mutants are very similar to those reported on inactivation of the KNR4 gene (Kapteyn et al., 1999
). This gene appears to encode a cytoplasmic protein that is part of a regulatory complex involved in the assembly of the cell wall (Martin et al., 1999
).
Although the mechanism underlying the above changes in cell wall polymers in GDA1 null mutants from both yeast species are not clearly understood, they may be related to changes in the O-glycosylation pathway. Recently, a highly O-glycosylated cell wall protein family that is not anchored to the wall by the clasical glycosylphosphatidylinositol anchor PIR-CWP was characterized (Toh-e et al., 1993; Mrsa and Tanner, 1999
). These proteins are thought to play a compensatory role when cell walls are weakened; O-linked sugars may be necessary to link these proteins to ß-1,3-glucans (Ketela et al., 1999
).Ccw11p, an abundant PIR-related protein, is absent in cell walls of a pmt4
strain, which is known to be defective in O-glycosylation (Mrsa et al., 1997
). Two potential sensors of cell wall stress, Hkr1p and Mid2p, are exclusively O-glycosylated (Yabe et al., 1996
; Kuranda and Robbins , 1991
) and may also play a role in regulating the different compensatory changes occurring in the wall of K. lactis and S. cerevisiae. The direct targets of these modifications will be subject of further studies and may provide insights into the molecular basis of the organization and dynamics of the yeast cell wall.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Isolation of KlGDA1 gene
A K. lactis genomic library previously made in our laboratory (Abeijon et al., 1996a) was screened at low stringency (Maniatis et al., 1997
) using the open reading frame (ORF) of the S. cerevisiae GDA1 gene as probe (Abeijon et al., 1993
). A positive clone, pDL-12, caused threefold overexpression of GDPase activity when transformed into K. lactis. Restriction analysis and sequencing of the 11.5-kb insert demonstrated a complete ORF of 1.5 kb that was homologous to the S. cerevisiae GDA1 gene. The whole ORF was located within a 1.9-kb HindIII fragment that was subcloned into the HindIII site of Kep6 to obtain pDL-13. The Kep6 vector has a segment of pKD1 that carries the replication origin of the 1.6-µm plasmid (Bianchi et al., 1987
). Stable replication of this vector requires the presence of resident pKD1 in the recipient yeast cell. The copy number and stability of this plasmid is comparable to the 2-µm plasmidderived vectors of S. cerevisiae. Kep6 also has the URA3 gene of S. cerevisiae that complements the uraA mutation of K. lactis; it also contains pBR322 sequences to allow selection and amplification in E. coli. The complete sequence of the 1.9-kb genomic HindIII fragment containing the KlGDA1 gene was deposited in the EMBL Nucleotide Sequence Database with the accession number AJ401304.
Construction of Klgda1 null mutant strain, KL8
Inactivation of the KlGDA1 gene was achieved by the URA blaster protocol (Alani et al., 1987). To obtain the disruption plasmid, pBlueKlhUh, the 1.9-kb HindIII fragment containing the ORF was cloned into the HindIII site of pBluescript (Stratagene, La Jolla, CA) whose BamHI site had been previously removed. An internal 0.9-kb BamHI fragment from the ORF was replaced with a 3.8-kb BamHIBamHl fragment from plasmid pNKY51 (kindly provided by N. Kleckner) that contained the URA3 gene flanked by hisG direct repeats. Recombination between the flanking direct repeats results in the elimination of the URA3 gene and recovery of uracil auxotrophy. Digestion of pBlueKlhUh with Xhol and EcoRl released a linear fragment containing the disruption cassette that was introduced in K. lactis strain K18 by electroporation. DNA from transformants was used to confirm the disruption of the KlGDA1 gene by polymerase chain reaction (PCR) analysis. The following primers were generated for this purpose: Forward primer 5'-CAAAAGCAAACAGCCAAAGACC-3' (located at position + 198). Reverse primer 5'-CGTCTTTGCTGTTCTCAACTCTC-3' (located at position +1475). These primers amplified a 1.2-kb PCR product in wild-type cells; in mutant cells a product of 4.3 kb was obtained. PCR of genomic DNA from mutant cells after the pop-out of the hisGURAhisG sequence resulted in a product of 1.6 kb. Correct integration was found in 1% of the clones and was further confirmed by assaying for reduced GDPase/UDPase activity in membrane protein extracts.
Construction of plasmids pKEpSC and pYEpKl for cross-species complementation
To express GDA1 and KlGDA1 in K. lactis and S. cerevisiae, we constructed the following plasmids. A 2.2-kb HindIIINheI fragment containing the S. cerevisiae gene was cloned into Kep6 previously opened with HindIII and NheI to generate the K. lactis plasmid pKEpSc. Similarly, a 1.9-kb HindIII fragment containing the KlGDA1 gene was cloned into the HindIII site of the S. cerevisiae plasmid Yep 352 to generate plasmid pYEpKl. K. lactis and S. cerevisiae null mutants were then transformed with pKEpSc and pYEpKl, respectively.
Preparation of vesicle fractions and sucrose gradient fractionation
Vesicle fractions were prepared from cells (6 L), grown to a density of 0.8 OD600. After washing with cold 10 mM sodium azide as previously described (Abeijon et al., 1993), cells were resuspended in spheroplast buffer (50 mM potassium phosphate, pH 7.5, 1.4 M sorbitol, 10 mM sodium azide, 0.3% ß-mercaptoethanol) containing 0.5 mg/ml of Zymolyase 100T (Seikagaku Co., Tokyo) and incubated at 37°C for 40 min. The spheroplast suspension was centrifuged and the pellet was washed with membrane buffer (10 mM triethanolamine acetic acid, pH 7.2, 0.8 M sorbitol, 1 mM EDTA, 1 µg/ml leupeptin, 0.5 mM PMSF) and resuspended in the same buffer. Spheroplasts were then broken by passage through a narrow-bore 10-ml glass pipette, diluted in membrane buffer, and centrifuged at 1000 x g for 10 min to obtain P1. The supernatant was centrifuged to 10,000 x g for 20 min to obtain P2, and the resulting supernatant was again centrifuged at 100,000 x g for 30 min to obtain P3. This fraction is enriched in Golgi markers, such as GDPase and
1,2 mannosyltransferase, and was used for nucleotide sugar transport assays. Vesicles were kept frozen at 70°C. Protein was measured using the BCA method (Bio-Rad, Hercules, CA).
For sucrose velocity gradient fractionation experiments, 1-L cultures were used. Speroplasts were prepared as described above and suspended in membrane buffer. The P2 supernatant fraction was prepared and the final concentration of MgCl2 was adjusted to 1 mM. Aliquots (9 ml) were placed on top of two 30-ml preformed, 2550% continuous sucrose gradients in Beckman SW28 centrifuge tubes. The sucrose solutions contained 1 mM MgCl2 and 10 mM triethanolamine acetic acid, pH 7.2. Gradients were centrifuged at 4°C for 90 min, at 25,000 r.p.m. in an L8-90 Beckman Ultracentrifuge as described previously (Abeijon et al., 1993). Twelve 2.5-ml fractions were collected from the top of each gradient, diluted, and centrifuged at 100,000 x g for 30 min; pellets were resuspended in membrane buffer.
Nucleotide diphosphate hydrolysis assay
Hydrolysis of GDP, UDP, and ADP was measured in vesicle fractions as previously described (Yanagisawa et al., 1990) in buffer containing 0.2 M imidazole, pH 7.5, 10 mM CaC12 or 10 mM MnC12, 0.1 % Triton X-100, and 2 mM GDP, UDP, or ADP. One hundred microliters of this solution, containing 510 µg of sample protein, were incubated at 30°C for 2030 min. The reaction was stopped by transferring the tubes to ice and adding 10 µl of 10% SDS. To determine the amount of phosphate released during hydrolysis, 200 µl of water and 700 µl of AMES reagent (1:6 mixture of 10% ascorbic acid and 0.42% ammonium molybdate in 1 N sulfuric acid) were added to each tube; following incubation at 40°C for 20 min, absorbance was measured at 660 nm.
1,2-N-acetylglucosaminyltransferase and
1,2-mannosyltransferase assays
Reactions were performed in a 50 µl final volume containing 30 µg of membrane protein, 50 mM HEPES, pH 7.2, 0.1% Triton X-100, 10 mM MnCl2, and 10200 µM GDP-[3H]mannose (0.1 µCi) or UDP-[3H]GlcNAc (0.1 µCi). As exogenous acceptors, 10 mM methyl D-mannopyranoside or 0.5 mM 3-O-
-D-mannopyranosyl-D-mannopyranoside were added for mannosyltransferase and GlcNAc transferase reactions, respectively. After, incubations at 30°C for 25 min, reactions were stopped by adding 0.4 ml of 10 mM EDTA. Radioactive substrates were separated from acceptors by binding of the former to a 1-ml Dowex-1 column; the radioactivity in the eluate, containing the acceptor, was measured. Between 10 and 60 units of potato apyrase (Grade VII, Sigma) were added to the reaction mixture when needed.
Nucleotide sugar translocation assay
The theoretical basis for the translocation assay of nucleotide derivatives into vesicles has been described in detail (Perez and Hirschberg, 1986). Briefly, Golgi-enriched vesicles (P3 fraction, 0.250.5 mg protein) were incubated at 30°C or 0°C for 3 min with the corresponding radioactive nucleotide sugar in the following 1-ml reaction mixture: 10 mM TrisHCl (pH 7.4), 0.25 M sucrose, 0.15 KCl, 1 mM CaC12, and 5 mM MgC12. Parallel incubations were done with the standard vesicle nonpenetrator [3 H] acetate. The reaction was stopped with 2 ml of ice-cold 0.25 M sucrose, 1 mM EDTA. Vesicles were separated from the incubation medium by centrifugation at 100,000 x g for 40 min. Total acid-soluble radioactivity, St, associated with the washed vesicle pellets was determined. The amount of radioactivity within vesicles in the pellet, Si, was calculated by multiplying the concentration of solutes in the incubation medium times the volume outside the vesicles in the pellet, Vo, of the nonpenetrator [3H] acetate (1.9 µl/mg protein). Transport activity is defined as Si after incubations at 30°C minus Si after incubations at 0°C. Latency of the vesicles was determined by measuring the lumenal Golgi GDPase activity, as described above, in the presence and absence of Triton X-100; it was 90% or higher in every instance. For gda1
strains, latency of N-actetylglucosamiyltransferase was measured.
Zymolyase sensitivity assay
Cells were grown to exponential phase at 23°C or 37°C. 5 x 108 cells were resuspended in 4 ml of buffered sorbitol (20 mM TrisHCl, pH 7.2, 1.2 M sorbitol, 10 mM MgCl2) with 3% 2-mercaptoethanol. After 10 min, 1 ml of 1 mg/ml Zymolyase 100T was added, and the cells were incubated at 23°C. Lysis was determined by measurements of A660 after 1:10 dilution in water of samples taken at 1.5-min intervals for K. lactis and 3-min intervals for S. cerevisiae, due to their different cell wall fragility.
Cell wall analysis
Cells were harvested and walls were isolated after mechanical disruption with glass beads (0.450.55 mm diameter) (as described in Perez and Hirschberg, 1986). Total alkali-insoluble fractions were obtained (Nguyen et al., 1998
) and ß-1,6/ß-1,3 glucan content was measured as described (Boone et al., 1990
). Hexose content was determined before and after dialysis using the borosulphuric acid method (Roemer and Bussey, 1991
). Chitin was determined according to Bulawa et al. (1986)
.
Chitinase analysis
Native chitinase was purified from late exponential cultures of K. lactis and S. cerevisiae mutant and wild-type cells grown in YPD. The S. cerevisiae mutant strain carrying the KlGDA1 gene was grown overnight in minimal medium and shifted to YPD for three generations. Chitinase was isolated by its binding to chitin as described (Maniatis, 1997). Proteins were then subjected to 6% SDSPAGE and detected by Western blot using a rabbit antiserum against chitinase (kindly provided by Dr. Charles Specht) and anti-rabbit IgG conjugated to peroxidase (Promega, Madison, WI). Final visualization was done with the Renaissance chemiluminescence detection kit (NEN Life Science, Boston, MA) according to the manufacturers instructions.
![]() |
Acknowledgments |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Abbreviations |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abeijon, C., Robbins, P.W., and Hirschberg, C.B. (1996b) Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc. Natl Acad. Sci. USA, 93, 59635968.
Abeijon, C., Yanagisawa, K., Mandon, E., Hausler, A., Moremen, K., Hirschberg, C.B., and Robbins, P.W. (1993) Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. J. Cell Biol., 122, 307323.[Abstract]
Alani, E., Cao, L., and Kleckner, N. (1987) A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics, 116, 541545.
Berninsone, P., and Hirschberg, C.B. (2000) Nucleotide sugar transporters of the golgi apparatus. Curr. Opin. Struct. Biol., 10, 542547.[ISI][Medline]
Berninsone, P., Miret, J.J., and Hirschberg, C.B. (1994) The Golgi guanosine diphosphatase is required for transport of GDP-mannose into the lumen of Saccharomyces cerevisiae Golgi vesicles. J. Biol. Chem., 269, 207211.
Bianchi, M.M., Falcone, C., Xin Jie, C., Weslowski-Louvel, M., Frontali, L., and Fukuhara, H.(1987) Transformation of the yeast Kluyveromyces lactis by new vectors derived from 1.6ÿm circular plasmid pKD1. Curr. Genet., 12, 185192.[ISI]
Boone, C., Sommer, S.S., Hensel, A., and Bussey, H. (1990) Yeast KRE genes provide evidence for a pathway of cell wall beta-glucan assembly. J. Cell Biol., 110, 18331843.[Abstract]
Brandan, E., and Fleischer, B. (1982) Orientation and role of nucleosidediphosphatase and 5"-nucleotidase in Golgi vesicles from rat liver. Biochemistry, 21, 46404645.[ISI][Medline]
Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, W.L. Jr., and Robbins, P.W. (1986) The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo. Cell, 46, 213225.[ISI][Medline]
Castro, O., Chen, L.Y., Parodi, A.J., and Abeijon, C. (1999) Uridine diphosphate-glucose transport into the endoplasmic reticulum of Saccharomyces cerevisiae: in vivo and in vitro evidence. Mol. Biol. Cell, 10, 10191030.
Gao, X.-D., Kaigorodov, V., and Jigami, Y. (1999) YND1, a homologue of GDA1, encodes membrane-bound apyrase required for Golgi N- and O-glycosylation in Saccharomyces cerevisiae. J. Biol. Chem., 274, 2145021456.
Guillen, E., Abeijon, C., and Hirschberg, C.B. (1999) Mammalian Golgi apparatus UDP-N-acetylglucosamine transporter: molecular cloning by phenotypic correction of a yeast mutant. J. Biol. Chem., 274, 66416646.
Hirschberg, C.B., Robbins, P.W., and Abeijon, C. (1998) Transporters of nucleotide sugars, ATP, and nucleotide sulfate in the endoplasmic reticulum and Golgi apparatus. Annu. Rev. Biochem., 67, 4969.[ISI][Medline]
Hong, Z., Mann, P., Brown, N.H., Tran, L.E. Shaw, K.J., Hare, P.S., and Di Domenico, B. (1994) Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis. Mol. Cell. Mol. Cell. Biol., 14, 10171025.[Abstract]
Kapteyn, J.C., van Egmond, P., Sievi, E., van Den Ende, H., Makarow, M., and Klis, F.M. (1999) The contribution of the O-glycosylated protein Pir2p/Hsp150 to the construction of the yeast cell wall in wild-type cells and beta 1,6-glucan-deficient mutants. Mol. Microbiol., 31, 18351844.[ISI][Medline]
Ketela, T., Green, R. and Bussey, H. (1999) Saccharomyces cerevisiae Mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway. J. Bact., 181, 33303340.
Kuranda, M., and Robbins, P.W. (1991) Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J. Biol. Chem., 266, 1975819767.
Maniatis, T., Fritsch, E.F., and Sambrook, J. (1997) Methods in yeast genetics: a laboratory manual. Plainview, NY: Cold Spring Harbor Laboratory Press.
Martin, H., Dagkessamanskia, A., Satchanska, G., Dallies, N., and Francois, J. (1999) KNR4, a suppressor of Saccharomyces cerevisiae cwh mutants, is involved in the transcriptional control of chitin synthase genes. Microbiology, 145, 249258.[Abstract]
Mrsa, V., and Tanner, V. (1999) Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast, 15, 813820.[ISI][Medline]
Mrsa, V., Seidl, T., Gentzsch, M., and Tanner, W. (1997) Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast, 13, 11451154.[ISI][Medline]
Nguyen, T.H., Fleet, G.H., and Rogers, P.L. (1998) Composition of the cell walls of several yeast species. Appl. Microbiol. Biotechnol., 50, 206221.[ISI][Medline]
Ohkubo, I., Ishibashi, T., Taniguchi, N., and Makita, A. (1980) Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein. Eur. J. Biochem., 112, 111118.[Abstract]
Perez, M., and Hirschberg, C.B. (1986) Topography of glycosylation reactions in the rough endoplasmic reticulum membrane. J. Biol. Chem., 261, 68226830.
Raschke, W.C., and Ballou, C.E. (1972) Characterization of a yeast mannan containing N-acetyl-D-glucosamine as an immunochemical determinant. Biochemistry, 11, 38073816.[ISI][Medline]
Roemer, T., and Bussey, H. (1991) Yeast beta-glucan synthesis: KRE6 encodes a predicted type II membrane protein required for glucan synthesis in vivo and for glucan synthase activity in vitro. Proc. Natl Acad. Sci. USA, 88, 1129511299.[Abstract]
Toh-e, A., Yasunaga, S., Nisogi, H., Tanaka, K., Oguchi, T., and Matsui, Y. (1993) Three yeast genes, PIR1, PIR2 and PIR3, containing internal tandem repeats, are related to each other, and PIR1 and PIR2 are required for tolerance to heat shock. Yeast, 9, 481494.[ISI][Medline]
Trombetta, E.S., and Helenius, A. (1999) Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum. EMBO J., 18, 32823292.
Wang, T.F., and Guidotti, G. (1998) Golgi localization and functional expression of human uridine diphosphatase. J. Biol. Chem., 273, 1139211399.
Wulff, C., Norambuena, L., and Orellana, A. (2000) GDP-fucose uptake into the Golgi apparatus during xyloglucan biosynthesis requires the activity of a transporter-like protein other than the UDP-glucose transporter. Plant Physiol., 122, 867877.
Yabe, T., Yamada-Okabe, T., Kasahara, S., Furuichi, Y., Nakajima, T., Ichishima, E., Arisawa, M., and Yamada-Okabe, H. (1996) HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae. J. Bact., 178, 477483.[Abstract]
Yanagisawa, K., Resnick, D., Abeijon, C., Robbins, P.W., and Hirschberg, C.B. (1990) A guanosine diphosphatase enriched in Golgi vesicles of Saccharomyces cerevisiae. Purification and characterization. J. Biol. Chem., 265, 1935119355.
Zhong, X., Malhotra, R., and Guidotti (2000) Regulation of Yeast Ectoapyrase Ynd1p Activity by Activator Subunit Vma13p of Vacuolar H+-ATPase. J. Biol. Chem., in press.