Global analysis of the glycoproteome in Saccharomyces cerevisiae reveals new roles for protein glycosylation in eukaryotes
Li A Kung1,ad, Sheng-Ce Tao2,3,4,bd, Jiang Qian5, Michael G Smith1, Michael Snyder1,c & Heng Zhu2,3
- Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, USA
- Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
- The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
- Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai, China
- Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA
Correspondence to: Michael Snyder1,c Department of Molecular, Cellular, and Developmental Biology, Yale University, PO Box 208103, New Haven, CT 06620-8103, USA. Tel.: +1 203 432 6139; Fax: +1 203 432 3597; Email: michael.snyder@yale.edu
Correspondence to: Heng Zhu2,3 Department of Pharmacology and Molecular Sciences and the HiT Center, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Tel.: +1 410 502 0878; Fax: +1 410 502 1872; Email: heng.zhu@jhmi.edu
Received 26 November 2008; Accepted 5 August 2009; Published online 15 September 2009
aPresent address: Helicos BioSciences, Building 700 3rd Floor, One Kendall Square, Cambridge, MA 02139, USA
bPresent address: Room 126, Wenxuan Building of Medicine, 800 Dongchuan Rd, Shanghai 200240, China
cPresent address: Department of Genetics, Stanford University, Stanford, CA 94305, USA
dThese authors contributed equally to this work
Top of pageArticle highlights
- We have developed a yeast proteome microarray based lectin competition binding assay for screening glycoproteins globally. To improve the coverage, both the two types of proteome microarray with either C-terminal tagged proteins or N-terminal tagged protein were used in this assay.
- We 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.
- We found 30 glycosylated proteins reside in the mitochondria. 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, Ydr065wp and Lpe10p, localization to the mitochondria is diminished upon tunicamycin treatment, indicating that protein glycosylation is important for protein function.
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; Dube and Bertozzi, 2005). 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).
Figure 1
A schematic representation of glycan competition assays. Proteome arrays fabricated with proteins of various types are probed with fluorescently labelled lectins. (Left track) A proteome chip is incubated with lectins, followed by a washing step to remove free lectins and some weak, non-specific interactions; however, stronger non-specific interactions, presumably caused by protein–protein interactions, still remain, leaving behind glycan-independent binding signals. (Right track) In parallel, a proteome chip is incubated with lectins in the presence of excess amount of glycan competitors, which should block glycan-dependent interactions. The comparison of signal intensities between the two tracks reveals proteins that show glycan-dependent binding activities.
Full figure and legend (170K)Figures & Tables indexAs 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). 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.
Figure 6
Effect of glycosylation on mitochondrial protein localization. (A) Localization of mitochondrial proteins, Ydr065wp and Lpe10p, is regulated by glycosylation. Yeast strains that harbour chromosomally GFP-tagged candidate genes were collected at the mid-log phase, incubated in the presence and absence of tunicamycin, and stained with MitoFluor Red 589 to visualize the mitochondria. In the untreated cells (Tunicamycin-), Ydr065wp and Lpe10p showed clear mitochondrial localization (GFP) and extensive overlapping staining with the MitoFlour (Mito and Merged). However, upon incubation with tunicamycin (Tunicamycin+), mitochondrial localization of both proteins is reduced and increased cytosolic localization is observed. (B) Quantitative measurement of mitochondrial localization with and without tunicamycin treatment. Cell counting was performed three times independently and 100 cells were counted for each strain in the presence and absence of tunicamycin treatment. The error bars represent the standard deviation.
Full figure and legend (262K)Figures & Tables indexOverall, 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.
Acknowledgements
We thank Dr Robert Jensen for help with yeast mitochondria staining. LK was supported by an NIH postdoctoral training fellowship. ST was supported by Shanghai Jiaotong University 985 grant. HZ was supported, in part, by NIH grants and the WW Smith Charitable Trust. MS was supported by NIH grants.
References
- Dube DH, Bertozzi CR (2005) Glycans in cancer and inflammation. Potential for therapeutics and diagnostics. Nat Rev Drug Discov 4: 477–488 | Article | PubMed | ISI | ChemPort |
- Helenius A, Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73: 1019–1049 | Article | PubMed | ISI | ChemPort |
