In this study, we have developed a lectin binding assay for identifying glycoproteins and used the assay to screen two types of the yeast proteome chips. These studies identified 534 yeast glycoproteins, 406 of them were novel and 45 of the novel glycoproteins were validated by gel shift. Combining our results with those of previous studies reveals a total of 350 validated glycoproteins, approximately 6% of the yeast proteome. This number likely is a minimum estimate since we only detected approximately 25% of known glycoproteins. Thus, we estimate that approximately 20–30% of yeast proteins are glycosylated, which is consistent with the previous estimation (Apweiler et al, 1999
Our validation focused on N
-linked glycosylation using EndoH and PNGase F, because dramatic changes can be observed in gel-shift assays. In principle, O
-linked glycosylation can be followed using endo-β-N
-acetylglucosaminidase F (Endo F), α-mannosidase, and O
-glycanase. However, O
-linked glycan chains are generally much shorter, and in yeast O
-modified proteins possess only a linear chain of up to five mannose residues (Kukuruzinska et al, 1987
); this makes validation with this approach more difficult.
The use of proteome chips is particularly well suited to the large-scale analysis of glycosylation for the following reasons: (1) the yeast proteins were purified from their original host that presumably preserves the native posttranslational modifications; (2) the native N- and C-termini ends of a given protein is likely to be preserved on the C- and N-terminal proteome chips, respectively; and (3) the use of the two types of chips likely increases the chances of detection; if a protein is failed to purify with a tag at one terminus, it has a second chance on the other proteome chip. Therefore, the use of two different types of chips should facilitate the identification of different types of glycosylations. This study used lectin probes for glycan detection rather than antibodies as was the case for a previous study (Gelperin et al, 2005
). The number of lectins with highly specific glycan-binding activity is much more than the available anti-glycan antibodies, which are difficult to generate with high specificity and their characterization is often limited (this was the case in the previous study). Although some lectins are known to carry non-specific binding properties, the addition of glycan inhibitors can dramatically increase the fidelity of the binding results, as we demonstrated in this study. Thus, the types of glycans can usually be detected using lectin probes.
Yeast has only two glycan modifications and is simple compared with other eukaryotes. Nonetheless, the wide spectra of glycan recognition by a large number of lectins (~100 are commercially available) should allow a similar strategy to be extended to higher eukaryotes, which have much more complicated glycan structures. Human protein chips containing over 8000 proteins are available (Invitrogen). Because of the larger numbers of possible modifications with different glycans (Gabius et al, 2004
), a larger number of lectins may be needed to determine which modifications are present on each protein. Analysis of these modifications is expected to provide insight into the ‘glycan code,' for determining the functional role of each type of glycan modification.
In addition to identification of proteins that reside or traverse secretory compartments, a variety of other types of proteins were found, including nuclear and mitochondrial proteins. The finding of a nuclear protein is new in yeast but not surprising in mammalian systems. Hart and co-workers have identified O
-linked glycosylation on nuclear and cytosolic proteins, including transcription factors, signalling components, and metabolic enzymes (Wells and Hart, 2003
). Further studies by Hart and others demonstrated that O
-linked glycosylation plays a role in modulating transcriptional activity, probably through control of protein stability, protein subcellular localization, and/or protein-O
–GlcNAc interaction. We find that the nuclear proteins that are modified are involved in diverse cellular processes (e.g., Sox2p). We speculate that the modification of these proteins by glycans might provide a mechanism for tying transcription control to metabolic state; the concentration of glycans might affect the amount of modified transcription factor and thus regulate the transcriptional activity of the cell.
The finding that several mitochondrial proteins are glycosylated was surprising. To date, only one mammalian glycoprotein in the mitochondria has been described in a single study (Chandra et al, 1998
). Additional mitochondrial proteins were found by Gelperin et al (2005)
, but the significance was not reported. Our finding of 30 mitochondrial glycoproteins, six of which were validated significantly, extends this list (). Four of these proteins (Ykl187Cp, Fks3p, Ydr065wp, and Ygl068wp) react with both ConA and WGA, suggesting they have both mannose and GlcNAc.
The fact that the mitochondrial protein glycosylation is blocked by tunicamycin suggests that these proteins either transverse the normal secretory pathway to reach the mitochondria, or that tunicamycin-sensitive enzyme(s) reside in the mitochondria. Evidence provided for both mechanisms (Rizzuto et al, 1998
; Bozidis et al, 2008
) suggest that the ER makes close contact and can exchange membranes with mitochondria in virally infected cells, suggesting that direct contact can occur. Evidence for modification enzymes in the mitochondria comes from Louisot and co-workers who demonstrated N
-linked glycoprotein synthesis occurs through dolichol intermediates in mammalian mitochondria, although they did not identify the protein substrates (Levrat et al, 1989
). The use of lipid-linked oligosaccharides as donors in N
-linked glycoprotein synthesis results in high-mannose structures that significantly differ from complex-type Asn-linked oligosaccharides in the plasma membrane. Perhaps, a similar, parallel N
-linked glycosylation system exists in the mitochondria in the budding yeast that is also tunicamycin sensitive.
Our finding of 30 mitochondrial glycoproteins reveals candidate substrates for the glycotransferases and indicates that a number of mitochondrial proteins are glycoproteins. Although previous studies have shown glycotransferase activity and identified a glycosylated protein in mammalian mitochondria (Chandra et al, 1998
), the biological relevance of such modifications remains elusive. Our study using tunicamycin inhibition demonstrates that glycosylation is important for the subcellular localization of at least two mitochondrial glycoproteins. Protein glycosylation has been shown to affect the distribution of proteins in the secretory pathway. Furthermore, Hart and co-workers showed that the balance between phosphorylation and glycosylation at Thr-58 regulated the distribution of c-Myc between the cytoplasm and the nucleus (Cole and Hart, 1999
). Thus, protein glycosylation may be a general mechanism for regulation of protein localization in a variety of cellular compartments.
In summary, we developed a new protein microarray strategy to globally identify glycoproteins in yeast and reveal new roles for protein glycosylation. Further definition and characterization of the glycome of yeast and other eukaryotes is expected to reveal additional novel roles or protein glycosylation in eukaryotes.