Cryo-EM structure of the E. coli translating ribosome in complex with SRP and its receptor

We report the 'early' conformation of the Escherichia coli signal recognition particle (SRP) and its receptor FtsY bound to the translating ribosome, as determined by cryo-EM. FtsY binds to the tetraloop of the SRP RNA, whereas the NG domains of the SRP protein and FtsY interact weakly in this conformation. Our results suggest that optimal positioning of the SRP RNA tetraloop and the Ffh NG domain leads to FtsY recruitment

Transport of Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na+ rather than H+. To elucidate the mechanism of Na+ transport, the C-terminally truncated NuoL subunit (NuoLN) which is related to Na+/H+ antiporters was expressed as a protein A fusion protein (ProtA-NuoLN) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA-NuoLN catalyzed the uptake of Na+ and K+ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA-NuoLN translocated Na+ and K+ independently from other complex I subunits. Na+ transport by ProtA-NuoLN was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na+/H+ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discusse

No-nonsense:insights into the functional interplay of nonsense-mediated mRNA decay factors

Nonsense-mediated messenger RNA decay (NMD) represents one of the main surveillance pathways used by eukaryotic cells to control the quality and abundance of mRNAs and to degrade viral RNA. NMD recognises mRNAs with a premature termination codon (PTC) and targets them to decay. Markers for a mRNA with a PTC, and thus NMD, are a long a 3′-untranslated region and the presence of an exon-junction complex (EJC) downstream of the stop codon. Here, we review our structural understanding of mammalian NMD factors and their functional interplay leading to a branched network of different interconnected but specialised mRNA decay pathways. We discuss recent insights into the potential impact of EJC composition on NMD pathway choice. We highlight the coexistence and function of different isoforms of up-frameshift protein 1 (UPF1) with an emphasis of their role at the endoplasmic reticulum and during stress, and the role of the paralogs UPF3B and UPF3A, underscoring that gene regulation by mammalian NMD is tightly controlled and context-dependent being conditional on developmental stage, tissue and cell types

Multi-level regulation of myotubularin-related protein-2 phosphatase activity by myotubularin-related protein-13/set-binding factor-2

Mutations in myotubularin-related protein-2 (MTMR2) or MTMR13/set-binding factor-2 (SBF2) genes are responsible for the severe autosomal recessive hereditary neuropathies, Charcot-Marie-Tooth disease (CMT) types 4B1 and 4B2, both characterized by reduced nerve conduction velocities, focally folded myelin sheaths and demyelination. MTMRs form a large family of conserved dual-specific phosphatases with enzymatically active and inactive members. We show that homodimeric active Mtmr2 interacts with homodimeric inactive Sbf2 in a tetrameric complex. This association dramatically increases the enzymatic activity of the complexed Mtmr2 towards phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. Mtmr2 and Sbf2 are considerably, but not completely, co-localized in the cellular cytoplasm. On membranes of large vesicles formed under hypo-osmotic conditions, Sbf2 favorably competes with Mtmr2 for binding sites. Our data are consistent with a model suggesting that, at a given cellular location, Mtmr2 phosphatase activity is highly regulated, being high in the Mtmr2/Sbf2 complex, moderate if Mtmr2 is not associated with Sbf2 or functionally blocked by competition through Sbf2 for membrane-binding site

Ribosome–SRP–FtsY cotranslational targeting complex in the closed state

The signal recognition particle (SRP)-dependent pathway is essential for correct targeting of proteins to the membrane and subsequent insertion in the membrane or secretion. In Escherichia coli, the SRP and its receptor FtsY bind to ribosome–nascent chain complexes with signal sequences and undergo a series of distinct conformational changes, which ensures accurate timing and fidelity of protein targeting. Initial recruitment of the SRP receptor FtsY to the SRP–RNC complex results in GTP-independent binding of the SRP–FtsY GTPases at the SRP RNA tetraloop. In the presence of GTP, a closed state is adopted by the SRP–FtsY complex. The cryo-EM structure of the closed state reveals an ordered SRP RNA and SRP M domain with a signal sequence-bound. Van der Waals interactions between the finger loop and ribosomal protein L24 lead to a constricted signal sequence-binding pocket possibly preventing premature release of the signal sequence. Conserved M-domain residues contact ribosomal RNA helices 24 and 59. The SRP–FtsY GTPases are detached from the RNA tetraloop and flexible, thus liberating the ribosomal exit site for binding of the translocation machinery