Characterization of TMEM209, a transmembrane protein of the nuclear envelope

by David Kohlhause
Doctoral thesis
Date of Examination:2024-12-12
Date of issue:2025-04-29
Advisor:Prof. Dr. Ralph H. Kehlenbach
Referee:Prof. Dr. Ralph H. Kehlenbach
Referee:Prof. Dr. Michael Thumm
Referee:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Henning Urlaub
Referee:Dr. Alexander Stein
Referee:Prof. Dr. Hauke Hillen
Persistent Address: http://dx.doi.org/10.53846/goediss-11229

 

 

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Abstract

English

The nuclear envelope (NE) comprises the outer nuclear membrane (ONM) and inner nuclear membrane (INM), which are connected at the level of nuclear pore complexes (NPCs). A variety of transmembrane proteins is found at the NE, fulfilling key functions such as nuclear integrity, chromatin organization and regulation of gene expression. TMEM209 is a transmembrane protein of the nuclear envelope containing two transmembrane domains (TMDs) in the N-terminal region. Despite its identification several years ago, this protein remains poorly characterized. Recent studies demonstrated TMEM209 interaction with Nup205, a component of the NPC. Moreover, a strong correlation was observed between V5-tagged TMEM209 and NPCs in mouse embryonic fibroblast cells. Furthermore, TMEM209 homologs have been suggested as core NPC components in Arabidopsis thaliana and Trypanosoma brucei, suggesting that TMEM209 might be a nucleoporin in human cells as well. In this work, several approaches were conducted to characterize TMEM209 in detail. Proximity mapping of TMEM209 was performed using RAPIDS (Rapamycin- and APEX-dependent identification of proteins by SILAC), which identified a variety of proteins at the INM (e.g. Lamin B1, emerin) and the NPC (e.g. Nup153, ELYS, Nup155). Proximity was further validated by proximity ligation assay (PLA). TMEM209 interaction partners were analyzed using co-immunoprecipitation (co-IP) experiments, revealing previously unidentified binding to the transmembrane nucleoporins POM121 and Nup210. To analyze the subcellular localization of TMEM209 at the nuclear envelope, super-resolution microscopy was performed, which showed a high co-localization with NPCs, supporting the hypothesis that TMEM209 is indeed a nucleoporin. Further analyses were performed to investigate the functional roles of TMEM209 at the NE. Overexpression of HA-TMEM209 strongly reduced the Nup210 intensity at the NPC. In addition, the deletion of 13 amino acids at the C-terminus of TMEM209 led to its aggregation at the NE, co-localizing exclusively with Nup210. Conversely, TMEM209 depletion did not alter expression levels of NE proteins, as analyzed by confocal microscopy and Western blotting. However, changes in the morphology of HeLa cells were observed following TMEM209 downregulation. Finally, flow cytometry analysis of synchronized cells in which TMEM209 was depleted revealed an impaired cell cycle, which predominantly occurred in S phase. To conclude, this comprehensive analysis enhances the understanding of TMEM209 at the nuclear envelope and suggests potential roles as a transmembrane nucleoporin.
Keywords: nuclear envelope; TMEM209; nuclear pore complex; inner nuclear membrane; cell cycle; nuclear envelope transmembrane proteins