Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states.

Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.

The expression of virulence determinants in Pseudomonas aeruginosa is coordinately regulated in response to both the social environment-commonly referred to as quorum sensing-and to environmental cues. In this study we have dissected the various independent regulation levels for pyocyanin production, which is influenced by the homoserine lactone-and Pseudomonas quinolone signal (PQS)-mediated quorum-sensing systems as well as by iron and phosphate availability. We demonstrate that the phosphate regulon is involved in the transcriptional activation of rhlR and the augmentation of PQS and pyocyanin production under phosphate limitation. However, we also observed an enhancement of rhlR transcription under low-iron medium conditions and after the addition of PQS that was independent of the phosphate regulon. These results highlight the complexity of secondary metabolite production in P. aeruginosa via environmental cues and the quorumsensing system.Bacterial organisms that elaborate traits tailored to their surroundings have better chances of surviving the pressures of unfavorable environmental conditions and host defenses. The outstanding capability of Pseudomonas aeruginosa for adaptation is reflected by the large number of putative transcriptional regulators (53), as bacterial differentiation is often controlled by transcription factors whose activity is influenced by local cues. Moreover, it has been recognized that environmental signals (33) as well as the social surrounding control bacterial virulence factor production. Cell-density-dependent gene regulation is commonly referred to as quorum sensing (QS) (20). QS is based on the release of soluble communicator molecules that trigger the transcription of QS-dependent genes when the bacterial population has reached a certain cell density. Many of these genes are involved in bacterial pathogenicity (9,19,39,40,50). Three chemically distinct signal molecules have been identified so far in P. aeruginosa. Two of these are acyl-homoserine lactones (AHL): a butyryl-homoserine lactone and a 3-oxo-dodecanoyl homoserine lactone, which together with their corresponding transcriptional activator proteins (R proteins) comprise the two hierarchically organized QS systems las and rhl (8,27,38,41,44) and control the expression of over 200 genes (23,48,57). The third signal molecule is 2-heptyl-3-hydroxy-4-quinolone (43). This Pseudomonas quinolone signal (PQS) interacts with the las and the rhl systems. While the las system seems to induce the production of PQS, exogenous PQS up-regulates the expression of the rhl system (15, 16, 31). In P. aeruginosa the impact of the rhl QS on the biosynthesis of the secondary metabolites pyocyanin and rhamnolipids is well documented (42). However, the production of these secondary metabolites also seems to be dependent on environmental cues (3,62). A link between QS and iron homeostasis was suggested previously (5,11,21,24,25,52,60). Moreover, PQS was shown to exhibit an iron-chelating activity, and PQS-dependent rhlR induction seems to be ...

Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phos-

Proteomics is required to provide insight into any type of subproteome. While the workflow based on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) can be applied for many subproteomes and comprises well-established strategies for data presentation and data analysis, the comprehensive investigation of membrane proteomes remains a challenging task. We present a number of procedures that provide an insight into such systems. We have established a novel protocol for the efficient preparation of membrane fractions, which is used here for the human pathogen Listeria monocytogenes that overcomes difficulties associated with ribosomes. Subsequently, we have used the combination of sodium dodecyl sulfate (SDS)-PAGE and liquid chromatography-tandem mass spectrometry for the characterization of the membrane proteome. Three hundred and one different membrane proteins could be identified, including 70 proteins that exhibited 2-15 transmembrane domains. However, a remarkably high ratio of proteins was detected in gel sections that were not in accordance with their expected migration behavior during SDS-PAGE. Protein identifications based on MASCOT significance criteria could be shown to be of high quality and therefore could not be the explanation of this observation. Consequently we have developed LaneSpector, a general visualization tool that allows the systematic comparison between apparent and calculated protein masses, which is routinely applicable to any high-throughput approach using a mass-dependent separation dimension prior to LC-MS/MS. The detailed presentation of the LaneSpector plot promotes the validation of the analytical process and might help to reveal relevant biological processes such as proteolysis or other post-translational modifications.