Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes

Abstract

To further understand the roles of protein glycosylation in eukaryotes, we globally identified glycan‐containing proteins in yeast. A fluorescent lectin binding assay was developed and used to screen protein microarrays containing over 5000 proteins purified from yeast. A total of 534 yeast proteins were identified that bound either Concanavalin A (ConA) or Wheat‐Germ Agglutinin (WGA); 406 of them were novel. Among the novel glycoproteins, 45 were validated by mobility shift upon treatment with EndoH and PNGase F, thereby extending the number of validated yeast glycoproteins to 350. In addition to many components of the secretory pathway, we identified other types of proteins, such as transcription factors and mitochondrial proteins. To further explore the role of glycosylation in mitochondrial function, the localization of four mitochondrial proteins was examined in the presence and absence of tunicamycin, an inhibitor of N ‐linked protein glycosylation. For two proteins, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through the cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization. ### Synopsis Protein glycosylation is ubiquitous in eukaryotes and has been implicated in a wide variety of biochemical and cellular processes, including protein folding, maintenance of cell structure, receptor–ligand interactions and cell signalling, cell–cell recognition, and defence ([Helenius and Aebi, 2004][1]; [Dube and Bertozzi, 2005][2]). In spite of its importance, both the number and different types of proteins that are glycosylated are not known, and thus it is likely that the full range of biochemical and cellular functions is not understood. Thus, systematic studies would be useful to learn more about the roles of glycosylation. We set out to globally identify glycoproteins in S. cerevisiae by probing the yeast proteome chips with lectins that recognize GlcNAc or mannose. To achieve this, we developed a glycan competition assay that allowed for sensitive detection of specific glycans by circumventing the non‐specific binding of ConA and WGA to the surfaces and ‘sticky’ protein spots while simultaneously maintaining strong signals. Also, to improve the coverage, both the two types of proteome microarray with either C‐terminal tagged or N‐terminal tagged protein were used in this assay ([Figure 1][3]). As a result, we greatly extended the number of known glycoproteins in yeast and identified several glycoproteins that localize to the mitochondrion. To further explore the function of glycosylation to these mitochondrial proteins, treatment of cells with an inhibitor of protein glycosylation disrupted the localization of two mitochondrial proteins were carried out (Ydr065wp and Lpe10p; [Figure 6][4]). In addition to providing a more comprehensive understanding of protein glycosylation in general, this study thus defines new roles for protein glycosylation in mitochondrial protein function and localization. Overall, our studies greatly extend our understanding of protein glycosylation in eukaryotes through cataloguing of glycoproteins, and describe a novel role for protein glycosylation in mitochondrial protein function and localization. Further definition and characterization of the glycome of yeast and other eukaryotes is expected to reveal additional novel roles or protein glycosylation in eukaryotes. Mol Syst Biol. 5: 308 [1]: #ref-19 [2]: #ref-12 [3]: #F1 [4]: #F6