␤-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent ␤-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent ␤-barrel OMPs largely via its N-domain but with ␤-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent ␤-barrel OMPs interact with SurA via their N-and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving ␤-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for ␤-barrel OMP biogenesis.

Secondary metabolites are usually the bioactive components of medicinal plants. The difference in the secondary metabolisms of closely related plant species and their hybrids has rarely been addressed. In this study, we conducted a holistic secondary metabolomics analysis of three medicinal Glycyrrhiza species (G. uralensis, G. glabra, and G. inflata), which are used as the popular herbal medicine licorice. The Glycyrrhiza species (genotype) for 95 batches of samples were identified by DNA barcodes of the internal transcribed spacer and trnV-ndhC regions, and the chemotypes were revealed by LC/UV- or LC/MS/MS-based quantitative analysis of 151 bioactive secondary metabolites, including 17 flavonoid glycosides, 24 saponins, and 110 free phenolic compounds. These compounds represented key products in the biosynthetic pathways of licorice. For the 76 homozygous samples, the three Glycyrrhiza species showed significant biosynthetic preferences, especially in coumarins, chalcones, isoflavanes, and flavonols. In total, 27 species-specific chemical markers were discovered. The 19 hybrid samples indicated that hybridization could remarkably alter the chemical composition and that the male parent contributed more to the offspring than the female parent did. This is hitherto the largest-scale targeted secondary metabolomics study of medicinal plants and the first report on uniparental inheritance in plant secondary metabolism. The results are valuable for biosynthesis, inheritance, and quality control studies of licorice and other medicinal plants.

Key words: Protein biogenesis, nascent polypeptides, -barrel outer membrane proteins, periplasmic proteins, transmembrane protein translocons, SecA, SecY During biogenesis, nascent polypeptides of many proteins have to be translocated across biological membranes by relying on specific protein-conducting channels. It remains a great challenge to unequivocally identify the specific membrane-integrated channel proteins that translocate particular client proteins in living cells. In Gram-negative bacteria, proteins destined to the periplasmic compartment or outer membrane are all synthesized in the cytoplasm and have to be translocated across the inner (i.e., the cytoplasmic) membrane. The currently prevailing perception is that all these transmembrane translocations occur by using the same SecY channel on the inner membrane. Nevertheless, this perception, formed largely based on genetic and in vitro studies, has not yet been proved by direct analysis in living cells. Here, mainly via a systematic in vivo protein photo-crosslinking analyses mediated by a genetically incorporated unnatural amino acid, we revealed that, in contrary to the long-held view, nascent polypeptides of β-barrel OMPs are not translocated across the inner membrane via the SecY channel, but through a shortened version of SecA, designated as SecA N , which exists as a membrane-integrated homo-oligomers. Furthermore, we demonstrated that SecA N is most likely part of the supercomplex that we revealed earlier as one which is responsible for the biogenesis of β-barrel OMPs in living cells and spans the cytoplasm, the inner membrane, the periplasm and the outer membrane.not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/121335 doi: bioRxiv preprint first posted online 2 IntroductionMany proteins have to be translocated across biological membranes during their biogenesis in both eukaryotic and prokaryotic cells (1-4). It has been revealed that such transmembrane translocations are commonly accomplished via the work of membrane-integrated protein-conducting channels (2,(4)(5)(6)(7). In Gram-negative bacteria, nascent polypeptides of both outer membrane proteins and periplasmic proteins, all being synthesized by the cytoplasmic ribosomes , are believed to be mainly translocated across the inner (i.e., cytoplasmic) membrane through the SecY protein-conducting channel present in the SecYEG translocon either in a co-or post-translational manner (2,6,7). In the outer membrane of Gram negative bacteria (as well as that of eukaryotic mitochondria and chloroplasts), the major type of proteins are the β-barrel outer membrane proteins (β-barrel OMPs), which primarily comprise β-sheets that adopt a unique, highly stable cylindrical, barrel-like topology, and function in a variety of biological processes (8,9). The nascent β-barrel OMPs, after crossing the inner membrane, will be further facilitated by protein factors located in the per...