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For determination of the nascent substrates of E. coli SRP, we used selective ribosome profiling (SeRP) (3) which compares two ribosome profiling data sets generated from the same culture (Extended [...], Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons (1). The existence of parallel co- and post-translational transport pathways (2), however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

Lipidomics of Golgi-derived COPI vesicles unravelled a partial segregation of cholesterol and most sphingomyelin species except for one (N-stearoyl sphingomyelin) from the vesicle fraction (5). To understand the molecular mechanisms [...], Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane (1). Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the head-group and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein (3,4), which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

Author(s): Tara Hessa [1, 4]; Nadja M. Meindl-Beinker [1, 4]; Andreas Bernsel [2, 4]; Hyun Kim [1]; Yoko Sato [1]; Mirjam Lerch-Bader [1]; IngMarie Nilsson [1]; Stephen H. White [3]; [...]

Author(s): Tara Hessa [1]; Hyun Kim [1]; Karl Bihlmaier [1, 4]; Carolina Lundin [1]; Jorrit Boekel [1]; Helena Andersson [3]; IngMarie Nilsson [1]; Stephen H. White [2]; Gunnar von Heijne [...]

Signal peptides have a short, positively charged amin-terminal n-region, a central hydrophobic h-region and a polar carboxy-terminal c-region. Many interactions between signal peptides and other molecules were believed to be hydrophobic in nature. Paetzel and colleagues have reported the crystal structure of a bacterial signal peptidase, revealing a new protease fold and a hydrophobic surface patch positioning the active site relative to lipid bilayers.