Interactors and inhibitors of the SecYEG translocon in E. coli

Medientyp: E-Book

Titel: Interactors and inhibitors of the SecYEG translocon in E. coli

Beteiligte: Oswald, Julia [Verfasser]; Koch, Hans-Georg [Akademischer Betreuer]; Koch, Hans-Georg [Sonstige]; Roussa, Eleni [Sonstige]

Körperschaft: Institut für Biochemie und Molekularbiologie ; Albert-Ludwigs-Universität Freiburg, Medizinische Fakultät

Erschienen: Freiburg: Universität, 2023

Umfang: Online-Ressource

Sprache: Englisch

DOI: 10.6094/UNIFR/238638

Identifikator:

Schlagwörter: Endoplasmatisches Retikulum ; Proteine ; Proteintransport ; Translokation ; Membrantransport ; Protein-Protein-Wechselwirkung ; (local)doctoralThesis

Entstehung:

Hochschulschrift: Dissertation, Universität Freiburg, 2023

Anmerkungen:

Beschreibung: Abstract: Under different environmental conditions, it is vital for bacteria to maintain a functional proteome. One of the adjusting screws is protein transport with the SecYEG translocon at its center. It coordinates the insertion of proteins into the membrane and the translocation of proteins across the membrane. The Sec translocon is a highly dynamic, modular protein complex with various assembly states and partner proteins, many of which interact with SecY only transiently and are therefore difficult to identify. In the future, it will be a major challenge to identify and functionally characterise all these partner proteins and to define the conditions under which these accessory proteins are vital for the function of the SecYEG translocon. One of these transient partner proteins is the yet uncharacterised protein YicN, which was found to co-purify with the SecYEG translocon. The present study aimed to explore YicN and its influence on the Sec translocon, protein transport, and the bacterial membrane. For this purpose, the effect of YicN overproduction on the integrity and composition of the cell membrane was observed, as well as the influence on protein transport in coupled transcription/translation in vitro systems. In vivo and in vitro cross-linking methods were used to study the interaction partners. YicN was found to be localised in the inner membrane of the E. coli cell and shows a PK-protected transmembrane domain as well as a periplasmic C-terminal loop. Its overproduction impaired the separation of the inner and outer membranes via sucrose-gradient centrifugation, as well as the membrane thickness and resistance to osmotic stress. Combined with the fact that YicN is placed under a σE promoter and is upregulated during biofilm formation, the interaction of YicN with the Sec translocon may cause inner to outer membrane stabilisation under outer membrane stress. Furthermore, the influence of YicN on the function of the SecYEG translocon was investigated: yicN overexpression impaired protein translocation, while the absence of YicN in the knock-out strain did not significantly change transport or insertion. Cross-linking data showed that YicN interacts with SecE, YidC, PpiD and YfgM, while direct cross-linking to SecY could not be observed so far. The function of the cross-links still has to be resolved and connected to the expression of yicN under stress conditions. An inhibitory assay was established to reduce SecY activity for further experiments with YicN but showed only small effects for Eeyarestatin 1 on protein translocation

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