Scientific Reports - Directed mutagenesis of large multi-subunit protein complexes by plasmid sub-fragmentation

In Mueller et al. the authors capture and visualize the native enzyme-substrate (Michaelis) and product complexes of nucleotide addition by bacterial RNA polymerase. Substrate coordination in a transition-state-like conformation, together with highly coordinated water molecules, suggests a positional (entropic) catalytic mechanism involving specific acid-base chemistry.

Nucleocytoplasmic large DNA viruses (NCLDVs) encode multi-subunit RNA polymerases (msRNAPs) that challenge conventional views of viral evolution. Phylogenetic and structural studies reveal that NCLDV ...

Cohesin is a multi-subunit molecular machine that is able to create lateral chromatin loops within a linear chromosome fiber. Despite intense study, a…

Cohesin is a multi-subunit molecular machine that is able to create lateral chromatin loops within a linear chromosome fiber. Despite intense study, a…

Pancreatic ductal adenocarcinoma (PDA) evades immune detection partly via autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I). Why MHC-I is susceptible to ...

Cohesin is a multi-subunit molecular machine that is able to create lateral chromatin loops within a linear chromosome fiber. Despite intense study, a…

Berquez et al. show that delayed ER exit of MHC class I in PDAC cells promotes its autophagic degradation via a TEX264-NBR1 complex. Impaired peptide loading increases MHC class I targeting to autophagosomes. A CRISPRi screen identifies the ER E3 ligase NFXL1, which promotes MHC class I ubiquitylation and autophagic capture.

The universally conserved Sec translocon and the YidC/Oxa1-type insertases mediate biogenesis of alpha-helical membrane proteins, but the molecular basis of their cooperation has remained disputed over decades. A recent discovery of a multi-subunit insertase in eukaryotes has raised the question about the architecture of the putative bacterial ortholog SecYEG-YidC and its functional mechanism. Here, we combine cryogenic electron microscopy with cell-free protein synthesis in nanodiscs to visualize biogenesis of the polytopic membrane protein NuoK, the subunit K of NADH-quinone oxidoreductase, that requires both SecYEG and YidC for insertion. We demonstrate that YidC is recruited to the back of the translocon at the late stage of the substrate insertion, in resemblance to the eukaryotic system, and in vivo experiments indicate that the complex assembly is vital for the cells. The nascent chain does not utilize the lateral gate of SecYEG, but enters the lipid membrane at the SecYE-YidC interface, with YidC being the primary insertase. SecYEG-YidC complex promotes folding of the nascent helices at the interface prior their insertion, so the examined cellular pathway follows the fundamental thermodynamic principles of membrane protein folding. Our data provide the first detailed insight on the elusive insertase machinery in the physiologically relevant environment, highlight the importance of the nascent chain for its assembly, and prove the evolutionary conservation of the gate-independent insertion route. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, Ke1879/3, 267205415 (CRC 1208) European Research Council, https://ror.org/0472cxd90, CRYOTRANSLATION

In Gram-negative bacteria, multi-subunit efflux pumps transport molecules across the inner membrane, periplasm, and outer membrane to the extracellular environment. These efflux pumps include the Resi...

The development of subunit vaccines that mimic the molecular complexity of attenuated vaccines has been limited by the difficulty of intracellular co-delivery of multiple chemically diverse payloads at controllable concentrations. We report on hierarchical hydrogel depots employing simple poly(propylene sulfone) homopolymers to enable ratiometric loading of a protein antigen and four physicochemically distinct adjuvants in a hierarchical manner. The optimized vaccine consisted of immunostimulants either adsorbed to or encapsulated within nanogels, which were capable of noncovalent anchoring to subcutaneous tissues. In female BALB/c and C57BL/6 mice, these 5-component nanogel vaccines demonstrated enhanced humoral and cell-mediated immune responses compared to formulations with standard single adjuvant and antigen pairing. The use of a single simple homopolymer capable of rapid and stable loading and intracellular delivery of diverse molecular cargoes holds promise for facile developmen

In Gram-negative bacteria, multi-subunit efflux pumps transport molecules across the inner membrane, periplasm, and outer membrane to the extracellular environment. These efflux pumps include the Resi...

In cells, multi-subunit coat protein complexes are responsible for coordinating the transport of transmembrane proteins and lipid cargo between membranes. At the endosome, cargo trafficking is medi…

The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we ...

Production of photosynthetic proteins encoded in the chloroplast genome depends on transcription by a multi-subunit RNA polymerase complex unique to the organelle. Here, we summarize insight from rec...

Prohibitins are multi-subunit protein complexes implicated in a variety of roles in mitochondria, including maintaining cristae architecture, regulating lipid metabolism, and mediating protein quality...

In cells, multi-subunit coat protein complexes are responsible for coordinating the transport of transmembrane proteins and lipid cargo between membranes. At the endosome, cargo trafficking is medi…