A proteomic approach including two-dimensional electrophoresis and MALDI-TOF analysis has been developed to identify the soluble proteins of the unicellular photosynthetic algae Chlamydomonas reinhardtii. We first described the partial 2D-picture of soluble proteome obtained from whole cells grown on acetate. Then we studied the effects of the exposure of these cells to 150 muM cadmium (Cd). The most drastic effect was the decrease in abundance of both large and small subunits of the ribulose-1,5-bisphosphate carboxylase/oxygenase, in correlation with several other enzymes involved in photosynthesis, Calvin cycle and chlorophyll biosynthesis. Other down-regulated processes were fatty acid biosynthesis, aminoacid and protein biosynthesis. On the other hand, proteins involved in glutathione synthesis, ATP metabolism, response to oxidative stress and protein folding were up-regulated in the presence of cadmium. In addition, we observed that most of the cadmium-sensitive proteins were also regulated via two major cellular thiol redox systems, thioredoxin and glutaredoxin.
The spatio-temporal organization of proteins within the cytoplasm of eukaryotic cells rests in part on the assembly of stable and transient multiprotein complexes. Here we examined the assembly of the multiaminoacyl-tRNA synthetase complex (MARS) in human cells. This complex contains nine aminoacyltRNA synthetases and three auxiliary proteins and is a hallmark of metazoan species. Isolation of the complexes has been performed by tandem affinity purification from human cells in culture. To understand the rules of assembly of this particle, expression of the three nonsynthetase components of MARS, p18, p38, and p43, was blocked by stable small interfering RNA silencing. The lack of these components was not lethal for the cells, but cell growth was slightly reduced. The residual complexes that could form in vivo in the absence of the auxiliary proteins were isolated by tandem affinity purification. From the repertoire of the subcomplexes that could be isolated, a comprehensive map of protein-protein interactions mediating complex assembly is deduced. The data are consistent with a structural role of the three nonsynthetase components of MARS, with p38 connecting two subcomplexes that may form in the absence of p38.Multiprotein complexes are molecular machines that are essential for organization of the proteome and for integration of cellular functions. Translation of genetic information involves several supramolecular assemblies, including the ribosome and multiprotein complexes involved in the initiation and elongation steps of the protein biosynthesis process (1). A complex containing the four subunits of elongation factor 1 (EF1A and the guanine nucleotide exchange factors EF1B␣, EF1B, and EF1B␥) and ValRS (valyl-tRNA synthetase) was described (2, 3). Several structural models of the ValRS-EF1A-guanine nucleotide exchange factor assembly have been proposed (2,4,5). This complex is believed to play a role in channeling of tRNA Val during translation (6). A multiaminoacyl-tRNA synthetase complex (MARS) 4 of about 1.5 MDa was described more than 20 years ago, but its physical and functional organization remain elusive (7). This complex is ubiquitous from Drosophila to mammals and contains the nine aminoacyl-tRNA synthetases ArgRS, AspRS, GlnRS, GluRS, IleRS, LeuRS, LysRS, MetRS, ProRS, and the three nonsynthetase components p18, p38, and p43 (8). Initial protein-protein interaction maps have been determined by using the yeast two-hybrid system (9, 10) or by in vitro cross-linking (11). A structural working model has been proposed by electron microscopy and three-dimensional reconstruction (12). The p38 component forms the platform for complex assembly (9, 13). A p38 deficiency is lethal in mice (14). Recent studies have indicated that some of the components of this complex lead a double life. They are essential components of translation when associated within MARS but may also play noncanonical functions after dissociation from the complex or following transport in other cellular compartments. LysRS associates wit...
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