Carbohydrate chains on yeast carboxypeptidase Y are phosphorylated
(glycoprotein high mannose oligosaccharides/nuclear magnetic resonance/a-mannosidase/phosphodiester)
CARL HASHIMOTO, ROBERT E. COHEN, WIE-JIE ZHANG, AND CLINTON E. BALLOU
Department of Biochemistry, University of California, Berkeley, California 94720
Contributed by Clinton Ballou, January 12, 1981
Carboxypeptidase Y, a vacuolar enzyme from Saccharomyces cerevisiae, was digested with endo-fi-N-acetyl-Dglucosaminidase H to release the four oligosaccharide chains that are linked to asparagine in the glycoprotein. The oligosaccharides were fractionated into a neutral and acidic component, and the latter proved to phosphorylated. From its gel filtration pattern, the neutral fraction was shown to be a mixture of at least four homologs, the smallest of which had a proton NMR spectrum almost identical to that given by an IgM oligosaccharide with eight mannoses and one N-acetylglucosamine [Cohen, R. E. & Ballou, C. E. (1980) Biochemistry 19, 4345-4358]. The yeast oligosaccharide has one additional mannose unit in an al-+3 or al-*6 linkage, whereas the larger homologs appear to have two, three, and four more mannose units. One phosphorylated oligosaccharide with a mannose/phosphate ratio of 12.5 was reduced with NaB3H4 and then subjected to mild acid hydrolysis. This released mannose and mannobiose that were glycosidically linked to the phosphate group, whereas complete acid hydrolysis yielded Dmannose 6-phosphate. The recovered oligosaccharide phosphomonoester, which contained 11 or 12 mannose units, was digested exhaustively with a-mannosidase, and the product of this reaction was treated with alkaline phosphatase, which yielded radioactive Man3GlcNAcH2. These results suggest that the mannosidase-resistant phosphorylated oligosaccharide has the structure Man– P–6aMan–aMan–6fBMan–s4GlcNAcH2, in which some of the phosphate groups are substituted with mannobiose instead ofmannose. A second phosphorylated oligosaccharide with a mannose/ phosphate ratio of 6.5 probably contains two phosphodiester groups, but its structure has not been investigated in detail.
Proc. Natl. Acad. Sci. USA Vol. 78, No. 4, pp. 2244-2248, April 1981.
The yeast Saccharomyces cerevisiae has extracellular periplasmic glycoproteins (mannoproteins), such as invertase, that contain =50% mannose (1) and intracellular glycoproteins, such as carboxypeptidase Y, that contain 15% mannose (2) and are similar in some respects to mammalian glycoproteins (3). The asparagine-linked carbohydrate chains of the external invertase contain 100-150 mannose units that are differentiated into a core unit of 11 mannoses and an outer chain of 90-140 mannoses (4). From the composition and number of the carbohydrate chains in carboxypeptidase Y, the average chain size appears to be -13 mannoses (5)-i.e., the size ofa core unit. From these facts, one might postulate that the externalization of a mannoprotein is associated with or dependent on the addition of the outer chain and that intracellular mannoproteins lack a signal that specifies outer-chain addition (6). Although it is an attractive idea, a strong argument against this hypothesis is our recent isolation of S. cerevisiae mutants that make and secrete mannoproteins, including invertase, that appear to possess only the core oligosaccharide units (7).
To determine whether the carbohydrate chains have any role in specifying the compartmentation of glycoproteins in yeast, we have undertaken a detailed comparison of the core oligosaccharide units from intracellular and extracellular mannoproteins. This preliminary report on the carbohydrate chains from carboxypeptidase Y shows that they are similar to oligosaccharides from mammalian glycoproteins and that some ofthe chains are phosphorylated. Because carboxypeptidase Y is found in the yeast vacuole (8), an organelle somewhat analogous to the lysosome of higher organisms, the phosphorylation of the carbohydrate chains may have a role in determining the localization of the enzyme, as it appears to have with some lysosomal enzymes (9).