Biology subjects

Stein, F.

Publications and source records attributed to Stein, F..

57 records · Page 4Linked to original sources

bioRxiv2019

Systematic localization of Gram-negative bacterial membrane proteins

The molecular architecture and function of the Gram-negative bacterial cell envelope is dictated by protein composition and localization. Proteins that localize to the inner (IM) and outer (OM) membranes of Gram-negative bacteria play critical and distinct roles in cellular physiology, however, approaches to systematically interrogate their distribution across both membranes and the soluble cell fraction are lacking. We employed multiplexed quantitative mass spectrometry to assess membrane protein localization in a proteome-wide fashion by separating IM and OM vesicles from exponentially growing E. coli K-12 cells on a sucrose density gradient. The migration patterns for >1600 proteins were classified in an unbiased manner, accurately recapitulating decades of knowledge in membrane protein localization in E. coli. For 559 proteins that are currently annotated as peripherally associated to the IM (Orfanoudaki and Economou, 2014) and display potential for dual localization to either the IM or cytoplasm, we could allocate 110 to the IM and 206 as soluble based on their fractionation patterns. In addition, we uncovered 63 cases, in which our data disagreed with current localization annotation in protein databases. For 42 of them, we were able to find supportive evidence for our localization findings in literature. We anticipate our systems-level analysis of the E. coli membrane proteome will serve as a useful reference dataset to query membrane protein localization, as well as provide a novel methodology to rapidly and systematically map membrane protein localization in more poorly characterized Gram-negative species.

microbiology↗

bioRxiv2019

The outer membrane lipoprotein NlpI nucleates hydrolases within peptidoglycan multi-enzyme complexes in Escherichia coli

The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh-like sacculus requires the combined activity of PG synthases and hydrolases. In Escherichia coli, the activity of the two bifunctional PG synthases is driven by lipoproteins anchored in the outer membrane. However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form multimeric complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and cell division biosynthetic complexes based on its localization and genetic interactions. In line with such a role, we reconstitute PG multi-enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that PG regulators and adaptors are part of PG biosynthetic multi-enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.\n\nSignificanceThe activity of PG hydrolases may cause lysis of the bacterial cell if left unregulated. Hence, the cell must have ways of regulating and coordinating their activities. Our current understanding of how this occurs is incomplete. In this work, we present the outer membrane (OM) anchored lipoprotein, NlpI, as a scaffold of peptidoglycan hydrolases. We propose that NlpI facilitates the formation of multi-enzyme complexes and that, along with other regulators, it coordinates a safe enlargement and separation of the PG layer in E. coli.

microbiology↗

bioRxiv2018

Effect of Sec61 interaction with Mpd1 on Endoplasmic Reticulum-Associated Degradation

Proteins that misfold in the endoplasmic reticulum (ER) are transported back to the cytosol for ER-associated degradation (ERAD). The Sec61 channel is one of the candidates for the retrograde transport conduit. Channel opening from the ER lumen must be triggered by ERAD factors and substrates. Here we identified new lumenal interaction partners of Sec61 by chemical crosslinking and mass spectrometry. In addition to known Sec61 interactors we detected ERAD factors including Cue1, Ubc6, Ubc7, Asi3, and Mpd1. We show that the CPY* ERAD factor Mpd1 binds to the lumenal Sec61 hinge region. Deletion of the Mpd1 binding site reduced the interaction between both proteins and caused an ERAD defect specific for CPY* without affecting protein import into the ER or ERAD of other substrates. Our data suggest that Mpd1 binding to Sec61 is a prerequisite for CPY* ERAD and confirm a role of Sec61 in ERAD of misfolded secretory proteins.

biochemistry↗