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. 2012 Oct;160(2):1037-51.
doi: 10.1104/pp.112.204263. Epub 2012 Aug 24.

Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics

Nino Nikolovski  1 , Denis Rubtsov, Marcelo P Segura, Godfrey P Miles, Tim J Stevens, Tom P J Dunkley, Sean Munro, Kathryn S Lilley, Paul Dupree
Affiliations

Putative glycosyltransferases and other plant Golgi apparatus proteins are revealed by LOPIT proteomics

Nino Nikolovski et al. Plant Physiol. 2012 Oct.

Abstract

The Golgi apparatus is the central organelle in the secretory pathway and plays key roles in glycosylation, protein sorting, and secretion in plants. Enzymes involved in the biosynthesis of complex polysaccharides, glycoproteins, and glycolipids are located in this organelle, but the majority of them remain uncharacterized. Here, we studied the Arabidopsis (Arabidopsis thaliana) membrane proteome with a focus on the Golgi apparatus using localization of organelle proteins by isotope tagging. By applying multivariate data analysis to a combined data set of two new and two previously published localization of organelle proteins by isotope tagging experiments, we identified the subcellular localization of 1,110 proteins with high confidence. These include 197 Golgi apparatus proteins, 79 of which have not been localized previously by a high-confidence method, as well as the localization of 304 endoplasmic reticulum and 208 plasma membrane proteins. Comparison of the hydrophobic domains of the localized proteins showed that the single-span transmembrane domains have unique properties in each organelle. Many of the novel Golgi-localized proteins belong to uncharacterized protein families. Structure-based homology analysis identified 12 putative Golgi glycosyltransferase (GT) families that have no functionally characterized members and, therefore, are not yet assigned to a Carbohydrate-Active Enzymes database GT family. The substantial numbers of these putative GTs lead us to estimate that the true number of plant Golgi GTs might be one-third above those currently annotated. Other newly identified proteins are likely to be involved in the transport and interconversion of nucleotide sugar substrates as well as polysaccharide and protein modification.

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Figures

Figure 1.
Figure 1.
LOPIT experimental design and data preprocessing workflow. Organelle separation is obtained by density gradient ultracentrifugation. Four LOPIT experiments were performed, each consisting of two four-plex iTRAQ labelings from seven density gradient fractions. One fraction is common in both four-plex labelings. The peptide abundance values from the MS/MS quantitation of the iTRAQ reporter ions are collapsed to a protein level for each four-plex labeling and are presented as color-coded values. The missing value imputation was performed on protein profiles that were present in at least two experiments.
Figure 2.
Figure 2.
Evaluation of the missing values imputation approach. The mean expected error of imputed data and its 95% confidence intervals (CI) are presented. The plot shows the results of 1,000 simulation runs of the imputation procedure.
Figure 3.
Figure 3.
Multivariate analysis of the LOPIT data. A, Principal component analysis (PCA) plot of 728 protein profiles from the LOPIT experiments 1 and 2. B, PCA plot of 717 protein profiles from the LOPIT experiments 3 and 4. C, PCA plot of the 1,385 protein profiles of the combined data sets (LOPIT experiments 1–4) with imputed missing values. The profiles of marker proteins with previously reported subcellular localization (training set proteins) are colored in the PCA plots based on the organelle of residence.
Figure 4.
Figure 4.
Analysis of the TMD characteristics of single-span membrane proteins classified as localized to the ER, Golgi/TGN, and PM. A, Mean hydrophobicity (GES scale) of the residues at each position along the aligned TMDs relative to the cytosolic edge. The GES hydrophobicity values represent the free energy of partitioning from water into a hydrophobic environment. B, Distribution of TMD lengths. The exoplasmic ends of the TMD were defined using the hydrophobicity-scanning algorithm as for the cytosolic ends. C, Mean values for the residue volume at each position along the TMDs. D, Cumulative distribution of the cytosolic extramembrane lengths. E, Analysis of the cumulative abundance of Leu, Phe, and Tyr along the TMDs. F, Analysis of the cumulative abundance of Ala, Ile, and Val along the TMDs.
Figure 5.
Figure 5.
Functional class assignments of the 197 Golgi-localized proteins. The classification is based on the TAIR annotation for the characterized proteins and the HHpred output predictions for the uncharacterized proteins, as indicated in Supplemental Table S2. The numbers of proteins per class are indicated in parentheses.
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