A quantitative liposome microarray to systematically characterize protein-lipid interactions.
Saliba, A.E., Vonkova, I., Ceschia, S., Findlay, G.M., Maeda, K., Tischer, C., Deghou, S., van Noort, V., Bork, P., Pawson, T., Ellenberg, J. & Gavin, A.C.
Nat Methods. 2014 Jan;11(1):47-50. doi: 10.1038/nmeth.2734. Epub 2013 Nov 24.
Lipids have a role in virtually all biological processes, acting as structural elements, scaffolds and signaling molecules, but they are still largely under-represented in known biological networks. Here we describe a liposome microarray-based assay (LiMA), a method that measures protein recruitment to membranes in a quantitative, automated, multiplexed and high-throughput manner.
Interactome map uncovers phosphatidylserine transport by oxysterol-binding proteins.
Maeda, K., Anand, K., Chiapparino, A., Kumar, A., Poletto, M., Kaksonen, M. & Gavin, A.C.
Nature. 2013 Sep 12;501(7466):257-61. doi: 10.1038/nature12430. Epub 2013 Aug 11.
The internal organization of eukaryotic cells into functionally specialized, membrane-delimited organelles of unique composition implies a need for active, regulated lipid transport. Phosphatidylserine (PS), for example, is synthesized in the endoplasmic reticulum and then preferentially associates--through mechanisms not fully elucidated--with the inner leaflet of the plasma membrane. Lipids can travel via transport vesicles. Alternatively, several protein families known as lipid-transfer proteins (LTPs) can extract a variety of specific lipids from biological membranes and transport them, within a hydrophobic pocket, through aqueous phases. Here we report the development of an integrated approach that combines protein fractionation and lipidomics to characterize the LTP-lipid complexes formed in vivo. We applied the procedure to 13 LTPs in the yeast Saccharomyces cerevisiae: the six Sec14 homology (Sfh) proteins and the seven oxysterol-binding homology (Osh) proteins. We found that Osh6 and Osh7 have an unexpected specificity for PS. In vivo, they participate in PS homeostasis and the transport of this lipid to the plasma membrane. The structure of Osh6 bound to PS reveals unique features that are conserved among other metazoan oxysterol-binding proteins (OSBPs) and are required for PS recognition. Our findings represent the first direct evidence, to our knowledge, for the non-vesicular transfer of PS from its site of biosynthesis (the endoplasmic reticulum) to its site of biological activity (the plasma membrane). We describe a new subfamily of OSBPs, including human ORP5 and ORP10, that transfer PS and propose new mechanisms of action for a protein family that is involved in several human pathologies such as cancer, dyslipidaemia and metabolic syndrome.
Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium.
van Noort, V., Seebacher, J., Bader, S., Mohammed, S., Vonkova, I., Betts, M.J., Kuhner, S., Kumar, R., Maier, T., O'Flaherty, M., Rybin, V., Schmeisky, A., Yus, E., Stulke, J., Serrano, L., Russell, R.B., Heck, A.J., Bork, P. & Gavin, A.C.
Mol Syst Biol. 2012 Feb 28;8:571. doi: 10.1038/msb.2012.4.
Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell.
Proteome organization in a genome-reduced bacterium.
Kuhner, S., van Noort, V., Betts, M.J., Leo-Macias, A., Batisse, C., Rode, M., Yamada, T., Maier, T., Bader, S., Beltran-Alvarez, P., Castano-Diez, D., Chen, W.H., Devos, D., Guell, M., Norambuena, T., Racke, I., Rybin, V., Schmidt, A., Yus, E., Aebersold, R., Herrmann, R., Bottcher, B., Frangakis, A.S., Russell, R.B., Serrano, L., Bork, P. & Gavin, A.C.
Science. 2009 Nov 27;326(5957):1235-40. doi: 10.1126/science.1176343.
The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification-mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.