Misfolded or unassembled membrane proteins in the endoplasmic reticulum (ER) are polyubiquitinated, translocated into the cytosol, and degraded by the proteasome, a poorly understood process that is c...

The authors use selective ribosome profiling to define how and when factors for N-glycosylation and membrane insertion engage and disengage from the core Sec61 translocation channel during biogenesis ...

Bacteria in the gut use an unusual molecular apparatus to insert complex proteins into their outer membrane.

By studying dynamic folding intermediates on the human ribosome, Pellowe et al. show that newly made domains help each other to fold but do not stably interact until synthesis is complete, avoiding in...

Leder et al. show that the chaperones PDIA6, Hsp70 BiP, ERdj3, PDIA1 and Hsp90 form co-condensates within the endoplasmic reticulum, enhancing folding and preventing misfolding of client proteins.

FoF1-ATP synthase converts proton motive force into ATP, with the H+/ATP ratio typically ranging from 2.7 to 5. Here, the authors engineered this enzyme to form multiple peripheral stalks, achieving a...

Defining the minimal genetic requirements for cellular life remains a fundamental question in biology. Genomic exploration continually reveals novel microbial lineages, often exhibiting extreme genome...

Cryo-electron tomography has emerged as powerful technology for in-cell structural biology, and in combination with breakthroughs in protein structure prediction, offers a unique opportunity for illum...

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