Determination of Genuine Residents of Plant Endomembrane Organelles using Isotope Tagging and Multivariate Statistics

The knowledge of the localization of proteins to a particular subcellular structure or organelle is an important step towardsassigning function to proteins predicted by genome-sequencing projects that have yet to be characterized. Moreover, the localizationof novel proteins to organelles also enhances our understanding of the functions of organelles. Many organelles cannot bepurified. In several cases where the degree of contamination by organelles with similar physical parameters to the organellebeing studied has gone unchecked, this has lead to the mis-localization of proteins. Recently, several techniques have emerged,which depend on characterization of the distribution pattern of organelles partially separated using density centrifugationby quantitative proteomics approaches. Here, we discuss one of these approaches, the localization of organelle proteins byisotope tagging (LOPIT) where the distribution patterns of organelles are assessed by measuring the relative abundance ofproteins between fractions along the length of density gradients using stable isotope-coded tags. The subcellular localizationsof proteins can be determined by comparing their distributions to those of previously localized proteins by assuming thatproteins that belong to the same organelle will cofractionate in density gradients. Analysis of distribution patterns canbe achieved by employing multivariate statistical methods such as principal component analysis and partial least squares discriminateanalysis. In this chapter, we focus on the use of the LOPIT technique in the assignment of membrane proteins to the plantGolgi apparatus and endoplasmic reticulum.