The secretory pathway in mammalian cells consists of an array of membrane-bounded organelles and transport carriers through which secretory proteins move in a stepwise fashion to reach their different cellular destinations. This arrangement means that biochemical operations carried out by the pathway, such as protein folding, protein glycosylation or lipid biosynthesis, can be compartmentalized, which enables efficient and specific reactions. Because of this link between subcellular localization and function, a quantitative map of the distribution of all the protein and lipid constituents of the secretory pathway (the secretome) is an essential first step to a comprehensive molecular understanding of how the pathway functions. For the past 30 years, cell biologists and biochemists have addressed this problem by imaging immunolabeled components in intact cells and by the biochemical analysis of subcellular fractions (reviewed in [1
]). Although these approaches have generated an enormous amount of detailed knowledge on the composition of the secretory pathway, the availability of the complete sequences of several eukaryotic genomes has recently enabled more systematic attempts to describe the secretory pathway comprehensively at the molecular level. Landmark studies in which a large, almost genome-wide, fraction of yeast open reading frames (ORFs) were tagged with green fluorescent protein (GFP) and their subcellular localizations determined in living cells have generated comprehensive localization maps for the Saccharomyces cerevisiae
and Schizosaccharomyces pombe
proteomes and thus also the secretory pathways in these organisms [3
]. Related approaches in mammalian cells have also been reported [5
], although these are still far less comprehensive than for yeast.
Subcellular fractionation followed by mass spectrometry (MS)-based analysis has also proved highly successful in mapping proteins to specific subcellular structures, such as the Golgi complex [7
] or clathrin-coated vesicles [10
]. Remarkably, recent advances in MS-based proteomics (reviewed in [12
]) now even allow estimation of the relative abundance of proteins in a specific biochemical fraction, opening up new avenues for defining a genome-wide localization map of a mammalian proteome. Taking advantage of this technological progress, Gilchrist and colleagues [13
] have recently produced an MS-based proteomic map of the major membrane-bounded entities of the mammalian secretory pathway - the endoplasmic reticulum (ER) and the Golgi complex. The significance of this well controlled piece of work is that it both complements and extends previous proteomic analyses of the mammalian secretory pathway [7
Gilchrist et al
] used classical biochemical procedures to isolate rough ER, smooth ER and Golgi membranes from rat liver, and then assessed fraction purity by electron microscopy and enzyme activity analyses. Solubilized membranes were subjected to gel electrophoresis and quantitative tandem MS, which identified peptides that could be mapped to more than 2,000 proteins. Assignment of these proteins to 23 different functional categories allowed the in silico
removal of 470 proteins that were probably contaminants, with almost two-thirds of these being residents of mitochondria and the plasma membrane. Throughout this study, independent samples were prepared and analyzed in triplicate, with principal coordinate analysis confirming that the ER and Golgi fractions were consistently distinct from one another. Further clustering of the identified proteins was facilitated by subfractionation using salt washing and Triton X-114 phase separation, which finally yielded an impressive list of 832 unique ER proteins, 193 proteins of the Golgi complex and COPI transport vesicles, and a further 405 proteins that were found in both fractions.
This seems to be the most comprehensive effort so far to elucidate the proteomes of the organelles of the mammalian secretory pathway. An impressive number of controls were incorporated at every step to give the highest degree of confidence in the lists obtained. Closer analysis of these lists reveals that the vast majority of well known residents of the organelles have been identified, for example the components of the protein folding and glycosylation machinery in the ER, and the protein-modification enzymes in the Golgi complex, in addition to more than 300 uncharacterized proteins. Of particular note is the identification of many cytoplasmic proteins that are only transiently associated with membranes, including many components of the actin and micro-tubule cytoskeletons. However, a significant number of likely contaminants also seem to be present in the fractions, highlighting the fact that, despite improvements in the sensitivity of MS, the limitation of this type of approach remains at the level of the organelle separation techniques. For example, the identification of proteins of the plasma membrane/endocytic machinery (such as the clathrin adaptor 2 alpha subunit (CALM) and the GTPase dynamin) in the ER fraction indicates the difficulty of separating these membranes.