Biosynthesis of Yeast Maman ISOLATION OF KLUYVEROMYCES LACTIS MANNAN MUTANTS AND A STUDY OF THE INCORPORATION OF N-ACETYL-D-GLUCOSAMINE INTO THE POLYSACCHARIDE SIDE CHAINS*

One side chain in the cell wall mannan of the yeast Kluyveromyces lactis has the structure (see article). (Raschke, W. C., and Ballou, C. E. (1972) Biochemistry 11, 3807). This (Man)4GNAc unit (the N-acetyl-D-glucosamine derivative of mannotetroase) and the (Man)4 side chain, aMan(1 yields 3)aMan(1 yields 2)aMan(1 yields 2)Man, are the principle immunochemical determinants on the cell surface. Two classes of mutants were obtained which lack the N-acetyl-D-glucosamine-containing determinant. The mannan of one class, designated mmnl, lacks both the (Man)4GNAc and (Man)4 side chains. Apparently, it has a defective alpha-1 yields 3-mannosyltransferase and the (Man)4 unit must be formed to serve as the acceptor before the alpha-1 yields 2-N-acetyl-glucosamine transferase can act. The other mutant class, mnn2, lacks only the (Man)4GNAc determinant and must be defective in adding N-acetylglucosamine to the mannotetrasose side chains. Two members of this class were obtained, one which still showed a wild type N-acetylglucosamine transferase activity in cell-free extracts and the other lacking it. They are allelic or tightly linked, and were designated mnn2-1 mnn2-2. Protoplast particles from the wild type cells catalyzed a Mn2+-dependent transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the mannotetraose side chain of endogenous acceptors. Exogenous mannotetraose also served as an acceptor in a Mn2+-dependent reaction and yielded (Man)4GNAc. Related oligosaccharides with terminal alpha (1 yields 3)mannosyl units were also good acceptors. The product from the reaction with alphaMan(1 yields 3)Man had the N-acetylglucosamine attached to the mannose unit at the reducing end, which supports the conclusion that the cell-free glycosyltransferase activity is identical with that involved in mannan synthesis. The reaction was inhibited by uridine diphosphate. Protoplast particles from the mmnl mutants showed wild type N-acetylglucosamine transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains. Particles from the mnn2-1 mutant failed to catalyze N-acetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent alpha-1 yields 2-N-acetylglucosamine transferase with kinetic properties similar to those of the wild type enzyme.

Protoplast particles from the mnnl mutants showed wild type N-acetylglucosamlne transferase activity with exogenous acceptor, but they had no endogenous activity because the endogenous mannan lacked acceptor side chains.
Particles from the mnn2-1 mutant failed to catalyze Nacetylglucosamine transfer. In contrast, particles from the mnn2-2 mutant were indistinguishable from wild type cells in their transferase activity. Some event accompanying cell breakage and assay of the mnn2-2 mutant allowed expression of a latent a-1 -+2-N-acetylglucosamlne transferase with kinetic properties similar to those of the wlld type enzyme.
Cell wall mannan-proteins of the yeasts Succharomyces cere-vi&e and Kluyveromyces la& have a linear (Y-l-+6-linked backbone to which oligosaccharide side chains are attached by (Y-1+2 and a-1+3 linkages (l-5) (Fig. 1). The large polymannose chains are attached, through N-acetyl-o-glucosamine, to asparagine units in the protein (6-8). Short mannooligosaccharides are also found linked 0-glycosidically to the hydroxyl groups of serine and threonine residues (6,8,9). These latter oligosaccharides have structures that are identical with the fragments produced by selective chemical cleavage of a-1+6 linkages in the large mannan chains (6). All of these side chains exhibit polymorphic structures that are strain-specific (10).
["C]Mannan synthesized endogenously from GDP-D-[WImannose by Saccharomyces carlsbergensis protoplast particles is a complex product of the action of several different mannosyltransferases (11). Attempts to study any of the transferases separately using cell wall mannan mutants of 8. ceretitie (12, 13) have thus far been unsuccessful. One handicap has been the relatively low activity of the mannosyltransferases involved in side chain synthesis (11,14).
The presence of N-acetyl-D-glucosamine in the side chains of K. Zactis mannan (5) offered the possibility to study transfer of a terminal sugar residue to endogenous mannan acceptors in the absence of synthesis of new mannosyl linkages. Using mannan mutants of K. la&s, we have demonstrated that addition of N-acetylglucosamine to the mannan side chains requires the presence of a precursor mannotetraose unit with a terminal (Y- structures of the polysaccharide components of the wild type mannans of (A) Saccharomyces cerevisiae X2180 and (B) Kluyveromyces lactis NRRL 1140. The latter shows only the outer chain components, and it has not been established that this mannan has an inner core structure similar tothat in S. cerevisiae mannan, although it does possess alkali-labile serineand threonine-linked oligosaccharides comparable to those in the outer chain. In this figure, M is n-mannose, P is phosphate, GNAc is hr-acetyl-n-glucosamine, whereas Ser, Thr, and Asn are amino acids in a protein chain. All anomeric linkages are (Y except for those in the trisaccharide connecting unit M+GNAc-+GNAc+ Asn which are all 0. The anomeric linkage of mannose to serine and threonine has not been established. endogenous mannotetraose side chain acceptors, thereby ruling out the requirement for new synthesis of this unit. The additional discovery that certain exogenous mannooligosaccharides are good acceptors in the cr-l-+2-N-acetylglucosamine transferase system has provided a convenient system with which to analyze the structural and regulatory gene defects of several K. Z&is mannan mutants. These strains, and mutants derived from them, were grown at 30" to late stationary phase in media containing 5% Dglucose, 0.5% yeast extract, and 0.3y0 Casamino acids. Mannan was extracted from cells by autoclaving them with 0.02 M sodium citrate, pH 7.0, and the extracted material was purified either by precipitation with Fehling's solution (28) or by-Cetavlon (hexadecyltrimethyl ammonium bromide) precipitation (6, 29). Purified mannan was acetolyzed as described by Raschke and Ballou (5).

Mild
Base Hydrolysis of Mannan-Mannan (2 g), purified by the Cetavlon nrocedure. was dissolved in 150 ml of 0.1 M NaOH and the mixtuie was allowed to stand at 23" for 24 hours. The solution was neutralized with 2 M acetic acid and dialyzed against 5 changes of 206 ml of water. The solution outside the dialysis bag, which contained the oligosaccharides released from linkage to serine and threonine, was lyophilized.

Methyl&ion
Procedure-Methylation of reduced oligosaccharide acetolysis products was performed by the Hakomori procedure (30). The methylsulfinyl anion was prepared according to Sanford and Conrad (31), whereas the methylation conditions followed those of Helleravist et al. (32). The reduced and methvlated oliaosaccharides were hydrolyzkd'as described by Raschke and Baliou (5). For analysis by gas chromatography-mass spectrometry, the hydrolysis products were converted to alditol acetates by reduction with sodium borohydride followed by acetylation with acetic anhydride and anhydrous sodium acetate (33) 1 Materials-Bio-Gel P-2 (200 to 400 and -400 mesh) and Dowex AG l-X2 (206 to 400 mesh) were obtained from Bio-Rad Laboratories. Descending paper chromatography was done on Whatman No. 1 filter paper using (in volume ratios) ethyl acetate-pyridine-water (5:3:2) (Solvent A) and ethyl acetate-pyridine-water-acetic acid (5: 5:3: 1) (Solvent B). Neutral sugars were detected with alkaline silver nitrate (23). Paper chromatograms were scanned for radioactivity by cutting a a-cm-wide strip into l-cm horizontal bands which were counted in 10 ml of Bray's solution (24) using a Packard Tri-Carb scintillation counter. B-n-Fructofuranosidase activity was assayed by a modification of the procedure of Bernfeld (25.26). Gas chromatography of partially methylated alditol acetates .was carried out a< 160' on a column (2.5 ft 3% OV-210) usine a Varian Aeroaranh 1400 instru-Antisera-Antiserum against K. lactis NRRL 1140. S. cerevisiae S288C and X2180, and KLeckera brevis 55-45 were prepared as reported earlier (34, 35). Antiserum specific for the N-acetylglucosamine-containing pentasaccharide side chain of K. lactis was prepared by adsorption of anti-K. lactis serum with S. cerevisiae X2180 cells (5, 34).

Isolation of Mannan
Mutants-Mutagenesis of K. lactis strains Y-43a hi.9 and Y-58a his4Cwith ethyl methanesulfonate was performed as described by Raschke et al. (12). The mutagenized cells were grown at 30" for 2 days, then harvested and resuspended in 0.9% NaCl. Wild type cells having the N-acetylglucosamine-containing pentasaccharide side chain were agglutinated with 0.5 ml of antiserum directed against that determinant.
After 1 hour, the suspension was shaken and the agglutinated cells were again allowed to settle. A portion, 0.2 ml, of the supernatant was added to 2 ml of fresh medium and grown at 30" for 48 hours. The agglutination and growth procedures were repeated twice. The resulting suspension, enriched in cells with altered surface determinants, was plated onto a complete agar medium. Following 48-hour growth, single colonies were selected and again streaked onto complete medium. After an additional 24 hours, the colonies were tested for their ability to agglutinate with antiserum against the N-acetylglucosamine-containing pentasaccharide of K. lactis bv the nrocedure of Antalis et al. (36) and mnn the re- The diploid stage of this yeast is transitory (27) and ascu~ disseccessive allele of a mutation leading to an alteration in mannan tion was performed after approximately 1% of the culture had structure.
formed zygotes and then sporulated.
Preparation of Protoplast Particles-Protoplasts were prepared by a procedure similar to that described by van Rijn et al. (37). K. lactis cells were grown to early log phase on 5oj, n-glucose, 0.5yo yeast extract, and 0.3% casamino acids, then harvested and washed with 1% KCI. Cells, 1 g wet weight, were incubated for 30 min at 30" in 10 ml of 0.05 M EDTA, pH 6.9, containing 10 mM dithiothreitol.
The treated cells were washed 3 times with lo-ml portions of 12% n-mannitol and suspended in 25 ml of 0.05 M sodium succinate, pH 5.8, containing 12% n-mannitol and 0.25 ml of Glusulase. After the digest had shaken gently at 30" for 30 to 60 min, only protoplasts were observed under a phase contrast microscope. The protoplasts were washed 3 times with 12yo n-mannitol by centrifugation and then were broken by addition of 0.1 Y imidazole-HCI, pH 6.5. The resulting particles were collected by centrifugation at 25,000 X q for 20 min, and the pellet was resuspended in 0.07 M imidazole-HCl, pH 6.5, containing 33% glycerol and stored at -10" usually for no more than 2 weeks before use.
Enzyme Assays with Endogenous Acceptors-Protoplast particles, 0.5 to 2.0 mg of protein, were incubated in assay mixtures containing 0.3 nmol of GDP-n-[UJ4C]mannose or 0.8 nmol of UDP-~-acetyl-n-[l-14C]glucosamine and 5 rmol of MnC12 in 0.05 Y imidazole-HCl, pH 6.5, in a final volume of 0.5 ml. The reactions were stopped by addition of 2 ml of absolute ethanol, the precipitate was collected by centrifugation, washed 4 times with 1 ml of ethanol, and dried. Selective acetolysis (1,28) was performed on the pelleted fraction, using 1 ml of a 1:l mixture of acetic anhydride and pyridine for the acetylation step and 2 ml of a 10: 1O:l mixture of acetic anhydride-acetic acid-concentrated sulfuric acid for the acetolysis step. The acetolysis products were deacetylated and separated by paper chromatography in Solvent A. Enzyme Assays with Exogenous Acceptors-Incubations were carried out as described above for endogenous mannan acceptors except that protoplast particles were preincubated 10 min both with and without oligosaccharide before addition of the UDP-Nacetyl-n-[1-Wlglucosamine to initiate the reactions. After a fixed reaction time, the mixture was applied to a column (0.5 X 6 cm) of Dowex AGl-X2 (200 to 400 mesh), prepared in a disposable Pasteur pipette, and allowed to flow into the bed of the column (approximately 2 min). The neutral material on the column was then eluted with 1 ml of water into a scintillation vial. A lo-ml portion of Bray's solution (24) was added to the vial and the sample was counted.  c Diluted anti-Kloeckera brevis serum that was capable of agglutinating S. cerevisiae X2180-1A mnnl cells which possess the a-n-mannosylphosphate determinant.

Selection of Kluyveromyces
la& Mannan Mutants-Two classes of mutants were found that failed to agglut,inate with K. la& antiserum directed against the (Man)4GNAc determinant (Table I). One class agglutinated with Succharomyces cerevisiae X2180 antiserum specific for the tetrasaccharide side chain, crMan(l-+3)aMan(l-+2)crMan( 1+2)Man, indicating that these mutants still made this unit. The other class of mutants failed to agglutinate with anti-X2180 serum, which suggested that they possessed side chains no longer than a trisaccharide (13). Neither the wild type strains nor the mutants reacted with antiserum directed against the cr+mannosylphosphate determinant (anti-Kloeckera bretis serum) (35). As expected, all strains failed to grow on a minimal medium unless supplemented with histidine.

Acetolysis of K. la&
Mutant Mannans-Acetolysis patterns of mannans from the wild type and one from each of the two classes of mutants are shown in Fig. 2. Acetolysis patterns of mannans from K. la& strains Y-43a(3-55) and Y-58a(54) lacked only the (Man)3GNAc and (Man)4GNAc fragments that are characteristic of mannans from the wild type parents. This suggested that these mutants were defective in the enzyme catalyzing transfer of N-acetylglucosamine onto the mannan side chains. The mutations were designated mnnl-1 for Y-43cu(3-55) and mnn-2 for Y-58a(54) after the genetic and biochemical analyses described below. Mannans from the K. lactis strains Y-43a(2-22) and Y-58a(lO) lacked not only the two above fragments but also the mannotetraose acetolysis product (Man)l. These were designated mnnl mutants and apparently were defective in an cr-1+3-mannosyltransferase analogous to the mnnl mutation first reported in S. cereuisiae (12). This result suggests that formation of the a-l-+3 linkage is required for attachment    of N-acetylglucosamine to the mannan side chains. The ratios of acetolysis fragments from several representative mutants are given in Table II. Methyl&ion Analysis of Acetolysis Fragments-- Table  III summarizes the results of analysis by gas chromatography-mass spectrometry of partially methylated alditol acetates prepared from the reduced acetolysis fragments of several mannans. Both the wild type Y-58a, and the mnn%8 mutant, Y-58a(54), produce similar mannotriose and mannotetraose fragments. The methylation results for the tetrasaccharides agreed with the structure crMan(l-+3)crMan(l-+Z)crMan(l+2)Man, whereas the trisaccharide fragments were a mixture of cyMan( 1+3)aMan-(1+2)Man and crMan(l+2)LuMan(l+2)Man in a 1:3 ratio. As expected for a mutant defective in the cr-1+3-mannosyltransferase (12), the reduced mannotriose fragment derived from Y-58a( 10) yielded no 1,3,5-tri-0-acetyl-2,4,6-tri-o-methylmannitol and must be exclusively 1+2-linked.
The oligosaccharides, which accounted for 5yo of the total mannan-protein carbohydrate, had the same chromatographic properties on Bio-Gel P-2 as the acetolysis fragments produced from Fehling'sprecipitated mannan of the corresponding strains. Several peaks resulted from base treatment of the wild type Y-58a mannan. Paper chromatography in Solvent A of each fragment was used to confirm their identities as mannose, mannobiose, mannotriose, mannotetraose, and (Man)4GNAc. Mild base treatment of mannan from the mnn.%-d mutant did not yield (Man)dGNAc, whereas mannan from the mnnl mutant lacked this oligosaccharide as well as (Man)(. The ratio of mannose to N-acetylglucosamine in the (Man)l-GNAc of the wild type was 4 as expected (5), whereas no Nacetylglucosamine was detected in the acetolysis fragments from either Y-58a(lO) or Y-58a(54). The mannose to phosphate ratio of the Y-58a mannan was 132, and was decreased slightly to 94 in the Y-58a(lO) mannan. The structural role of phosphate in K. l&is mannan has not been investigated.

&Fructojuranosidase-Content
and Glusulase-Sensitivity of K. la& Mutants--Cell wall mannan mutants of S. ccre&ioc (12, 13) have different capacities to retain external invertase when whole cells are treated with mercaptans (38). In contrast, stationary phase K. la&s wild type and mutant cells released similar amounts (8 to 10%) of their external invertase into the medium on incubation for 150 min with 10 mM dithiothreitol. However, alteration of the cell wall structure of the K. Zactis mutants was apparent from the differential sensitivities of the mutant and wild type cells to ensymic lysis in a hypotonic buffer containing Glusulase. Removal of terminal N-acetylglucosamine and mannose units from the mannan side chains apparently made the cell wall glucan more susceptible to glucanases. Mild Base Hydrolysis Products of K. lactis Mannans-In con-Segregation of Mutant Phenotypes-Evidence that the K. lactic trast to mannan prepared by the Fehling's procedure, mannan strains with altered mannans were the result of single mutations purified by Cetavlon precipitation yielded a product with intact was obtained by tetrad analysis of crosses between wild type and glycosylserine linkages, and mild base treatment of such mannan mutant strains (39,40). All crosses yielded tetrads with viable released these oligosaccharides (6). Fig. 3 shows the products spores in which the mutant mannan phenotype, scored by ag- glutination with antisera directed against the (Man)4 and (Man)4GNAc determinants, segregated 2+:2-. About 8 tetrads were analyzed from each cross.
Various crosses of mnnl , mnnd-1, and mnn%'-8 mutants yielded the results summarized in Table IV. Independently derived mnnl mutations from the a and LY mating types were closely linked as shown by the formation only of parental ditype tetrads when the two identical phenotypes were crossed. The same was true for mnnl-1 mutants. These data suggest that the nonsegregating mutant pairs were allelic (40). The diploid from a cross of Y-43a  with Y-58a(54) yielded only parental ditype tetrads although these two mutants differ biochemically as shown below. Thus, these two mutants also appear to be allelic, although some difficulties in genetic studies with this yeast make the results somewhat unreliable.
Crosses of mnnl with mnn,%l and mnn.%~ mutants gave segregation frequencies characteristic of unlinked genes. In addition, the tetratype frequency was that expected if either the mnnl gene or the mnn2 gene were not centromere linked. As a control, tetrads of all crosses were examined for segregation of the unlinked histidine markers present in the two parents. His3 by hk4C crosses had a PD :NPD:T ratio of 15:10:62, close to the 1: 1:4 ratio expected for these unlinked genes that are not centromere linked (27, 40). Incorporation of N-Acetylglucosamine into Endogenous Mannan Acceptors-Incubation of broken protoplast preparations from the wild type K. Zactis Y-58a with UDP-N-acetyl[l-14C]glucosamine led to incorporation of radioactivity into ethanolinsoluble material. Treatment of the ethanol-precipitated product with 0.1 M NaOH for 24 hours did not release any 14C-labeled oligosaccharide that chromatographed in Solvent A with (Man)4GNAc.
Thus, the glucosamine had not been transferred to serine-linked oligosaccharides.
In contrast, the mild baselabile products accounted for over 60% of the radioactivity incorporated with GDP[W]mannose as the glycosyl donor. Partial acetolysis of the ethanol-insoluble material yielded radioactive products with the mobilities of (Man)3GNAc and (Man)4GNAc (Fig. 4). (Man)3GNAc normally results from degradation of (Man)eGNAc during acetolysis (5). Fig. 5 shows the gel filtration properties of the radioactive acetolysis fragment which chromatographed with (Man)4GNAc in Solvent A. The radioactivity was eluted in the same position as (Man)4GNAc produced by acetolysis of K. lactis Y-58a mannan. as chitin (41), was obtained in the ethanol-insoluble material in the absence of Mn*+ (but in the presence of Mg*+), no acetolysis product with a mobility of (Man)4GNAc was detected under these conditions. Optimum synthesis occurred with 10 mM Mn2+, and half-maximal activity occurred with a Mn*+ concentration of 2.5 mM. This Mn*+ requirement is similar to that reported for mannan biosynthesis from GDP-mannose (11,14). Preincubation of the protoplast particles with GDP-n-mannose did not increase their acceptor activity on addition of UDP-N-acetyl-[ l-14C]glucosamine, which suggests that new mannan synthesis was not required for incorporation of N-acetylglucosa-3431  mine. This was confirmed by the very low incorporation of mannose from GDP-D-[ U-"Clmannose into the endogenous acceptor of the same particles.
Protoplast particles, prepared from K. Zuctis wild type and mutant strains of each mating type, were incubated with M& and UDP-N-acetyl-[lJ4C]glucosamine.
The rate of formation of (Man)rGNAc-14C, isolated by acetolysis of endogenous ethanolinsoluble product, is shown in Table V. As expected, both wild type strains yielded (Man)'GNAc whereas mnnd-1 mutants failed to produce this fragment. Since the mannan of Y%a(54) lacks the (Man)lGNAc side chain, and this mutant and Y-43~ (3-55) appear to be allelic, it was surprising that the mnn$?-d mutant of K. la&is Y-58a did yield substantial amounts of (Man)'GNAc.
As shown below, this difference between the mn& mutants was also observed with exogenous mannooligosaccharide acceptors.
Incorporation of N-Acetylglucosamine into Exogenous Oligosacchuride Acceptors-Incubation of the tetrasaccharide, cyMan-(1+3)cuMan(l-+2)aMan(l-+2)Man, with K. la& Y58a protoplast particles and UDP-N-acetyl-[lJ4C]glucosamine in the presence of MnZ+ led to a dramatic increase in the amount of neutral radioactivity that could be eluted following Dowex-1 chromatography of the total reaction mixture (Fig. 6). Incorporation of radioactivity into other oligosaccharides also occurred, but only with those acceptors that contained a nonreducing terminal cr-1-3~linked mannose unit (Table VI) neither 3-0-methylmannose nor the pentasaccharide, aMan-~1--+3)cYMan(l+3)cuMan(l-+2)cYMan(l+2)Man (17), served as a substrate for the N-acetylglucosamine transfer. Fig. 7 shows the chromatographic behavior of the radioactive products resulting from incubation of Y-58a particles, UDP-N-acetyl-  the rate of soluble neutral product formation w&s determined ae described in the text. A minimum of 3 time points was used for each rate determination.
[ I-lC]glucosamine and Mn*+ with ruMan(l-+3)Man, crMan-(1+3)aMan(l+2)Man, and cuMan(l+3)crMan(l+2)cYMan-(1+2)Man. The products had mobilities expected for mannooligosaccharide substrates to which a single N-acetylglucosamine residue had been attached. The incorporation of N-acetylglucosamine from UDP-N-acetylglucosamine into soluble oligosaccharides showed a Mn*+-dependence similar to that for incorporation into the endogenous mannan. Uridine diphosphate at 1 mM gave complete inhibition of a reaction mixture synthesizing (Man)'-GNAc from (Man)'. Approximately 50% inhibition was observed with 0.1 mM UDP, a concentration at which UMP, GDP, and GMP caused less than a 10% decrease in the rate of N-acetylglucosamine transfer. We did not attempt to prepare lipid-free protoplast particles to test for a lipid requirement, but we did find that a lipid extract of yeast cells failed to stimulate the activity of the particulate glucosamine transferase. GDP-mannose did not inhibit the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine as might be expected were the same lipid involved in the transfer of mannose to mannan acceptors.
As expected, formation of (Man)aGNAc from (Man)' was catalyzed by protoplast particles from Y-58a, Y-43a, and both mnnl mutants (Table VII), but not by particles from mnni?-1 mutants. However, consistent with the results obtained with endogenous mannan acceptors, the mnn&d mutant derived from Y-58a did form (Man)rGNAc from exogenous (Man)'. This latter reaction showed a Mn*+-dependence identical to that observed with the parent strain and the K, for (Man)' (13 mM) was also similar to that observed with Y-58a protoplast particles (Fig. 8). Furthermore, the rate of (Man)'GNAc formation by Y-58a and Y-58a(54) particles increased similarly in response to increasing UDP-N-acetylglucosamine concentrations over the range of lOmE to 10-l M.
The failure of the Y-58a(54) mutant to produce (Man)dGNAc side chains in the intact cell could have been due to the overproduction of some inhibitor. Glucosamine transferase assays on the whole cell extract of the mutant did show about one-third of the total activity of the isolated protoplast particles, but the wild type extracts showed a similar reduction in activity. This effect was eventually found to result from a stimulation of the transferase activity by glycerol that was added to the resuspended particles but that was absent in the broken protoplast preparation that contained the whole cell extract. The possibility also existed that the glucosamine transferase in the mutant occurred in an inactive form that was activated by nonspecific proteases following cell breakage. However, we were not able to isolate such an inactive zymogen by cell disruption at low temperature or in the presence of protease inhibitors such as phenylmethane sulfonyl fluoride.
Characterization of Product Formed with cuMan(l -@Man as Acceptor-The disaccharide acceptor, 8 pmol, was incubated with 1.6 nmol of UDP-N-acetyl-[lJ4C]glucosamine, 5 pmol of MnC12, and protoplast particles (2 mg of protein) from K. Zactis Y43a! in a final volume of 0.5 ml of 0.05 M imidazole-HCl buffer, pH 6.5, for 1 hour at 30". The reaction mixture was applied directly to a column (0.5 x 6 cm) of Dowex AG l-X2 to remove excess radioactive UDP-N-acetylglucosamine, and the material eluted from the column with water was applied to a column (1 x 100 cm) of Bio-Gel P-2. The position of elution of the radioactive product corresponded to that of a tetrasaccharide ( Fig.  9) which would be expected for a compound with two mannose units and one N-acetylglucosamine (42).
The radioactive fractions were pooled and concentrated to 0.2 ml on the rotary evaporator. A 50-~1 sample was hydrolyzed in 1 N HCl at 100' for 3 hours, the acid was removed by evaporation under vacuum, and the residue was reduced with sodium borotritide at PH 10. The excess reducing agent was destroyed by adding acetic acid, and the boric acid was removed by repeated evaporation of the sample after addition of methanol. Paper chromatography of this hydrolyzed and reduced product (Solvent B) revealed the presence of mannitol, reduced N-acetylglucosamine and reduced glucosamine (Fig. 10). The latter was presumably formed by de-N-acetylation during the acid hydrolysis. Thus, the product of the above enzymic reaction contained mannose and N-acetylglucosamine.
A second portion of the NaBT4-reduced product was partially hydrolyzed with 0.3 N HCl at 100" for 2 hours. After neutralization of the reaction with dilute NaOH, the product was applied to a column (1 x 100 cm) of Bio-Gel P-2 and resolved by elution with water into two radioactive substances, one with the size of the starting material and the second approximately the size of a trisaccharide (Fig. 11). This product was presumed to be a disaccharide composed of one mannose unit and one N-3433 A, N-acetylglucosamine; B, reduced N-acetylglucosamine; C, mannose; D, mannitol; E, glucosamine; F, reduced glucosamine. acetylglucosamine.
The substance was labeled both with "C and "H, indicating that the N-acetylglucosamine had been added to the mannose at the reducing end of the disaccharide acceptor. To confirm this conclusion, the product from partial acid hydrolysis was subjected to complete hydrolysis in 1 N HCl at 100" for 3 hours and the hydrolysate was chromatographed on paper with Solvent B. Radioactive peaks corresponding to mannitol, N-acetylglucosamine and glucosamine were obtained. All of the above results demonstrate that N-acetylglucosamine was transferred to  expected if the cell-free reaction had the same specificity as the transferase of the intact cell that adds N-acetylglucosamine to the corresponding position of the mannotetraose side chain.

DISCUSSION
Yeast mannan biosynthesis appears to involve at least four levels of glycosylation; namely, those reactions for synthesis of the serine-and threonine-linked units (6, 9), for synthesis of the inner core (8), for synthesis of the outer chain (ll), and for addition of the substituents such as mannosylphosphate and Nacetylglucosamine that modify the outer chain (l-5).
In this study we have dealt with the last kind of reaction in which Nacetyl-n-glucosamine is added in a-l +2 linkage to mannotetraose side chains during the maturation of Kluyveromyces la& mannan. This reaction occurred with both endogenous and exogenous acceptors, using protoplast particles from the wild type strain of K. la&, with UDP-N-acetyl-n-[l-14C]glucosamine as the donor. The endogenous reaction product yielded (Man)aGNAc on acetolysis, whereas the exogenous reaction with (Man)p as the acceptor yielded (Man)4GNAc directly. Di-, tri-, and tetrasaccharides of mannose with a single terminal a-1 -+3 linkage served as acceptors, but a pentasaccharide with two terminal a-1-+3 linkages was inactive. In this reaction, the N-acetylglucosamine was added to the mannose unit at the reducing end of the disaccharide acceptor aMan(l+3)Man, a result consistent with the location of this amino sugar in the tetrasaccharide side chains of the mannan (5). Thus, the N-acetylglucosamine transferase is quite specific even though it appears to be involved in adding this sugar to those mannotetraose units both in the outer chain and on serine and threonine. The reaction was inhibited by uridine diphosphate in a manner suggesting that a lipid-P-GNAc intermediate could be involved, but alternative explanations for this inhibition are possible. Because the N-acetylglucosamine was added to endogenous mannan side chains in the absence of synthesis of new mannosyl linkages, the reaction probably proceeds by a stepwise addition and not by polymerization of preformed oligosaccharide side chains linked to lipid. The a-1 +2-N-acetylglucosamine transferase activity is similar in its Mn*f-dependence (11, 14) and nucleotide diphosphate inhibition (43) to the mannosyltransferase studied previously (43)(44)(45)(46)(47). Lehle and Tanner (48) report that