Characterization of VAPA at the inner nuclear membrane: localization, membrane insertion and nuclear interactome
von Inés Rodríguez González
Datum der mündl. Prüfung:2024-08-06
Erschienen:2024-12-19
Betreuer:Prof. Dr. Ralph Kehlenbach
Gutachter:Prof. Dr. Ralph Kehlenbach
Gutachter:Prof. Dr. Heike Krebber
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-10938
Dateien
Name:Dissertation_InesRodriguezGonzalez_Corrected.pdf
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Description:Final Thesis Corrected
Diese Datei ist bis 05.08.2025 gesperrt.
Zusammenfassung
Englisch
Tail-anchored (TA) proteins are type IV single-pass membrane proteins characterized by a long hydrophobic transmembrane domain (TMD) positioned very close to a notably short C-terminus facing the lumen. Due to their topology, most TA proteins require post-translational membrane insertion mechanisms since their TMD only emerges from the ribosome after conclusion of their translation. In humans, hundreds of TA proteins populate the membranes of different organelles regulating a plethora of cellular functions. This study mainly focuses on the TA protein VAPA. VAPA (Vesicle-associated membrane protein-Associated protein A) is an endoplasmic reticulum (ER) resident integral membrane protein involved in building the majority of membrane contact sites between the ER and several organelles, alongside its homologue VAPB. Both proteins are very similar in sequence and domain organization, and analyses of their interactomes have shown multiple shared interaction partners. However, their interactomes do not fully overlap, suggesting distinct and specific roles for each protein. In addition to the ER, VAPB has been identified to also localize at the inner nuclear membrane (INM). Thus, the primary objectives of this study were to analyze whether VAPA could reach the INM as well, to perform its proximity mapping and subsequently identify possible nuclear interaction partners. Its membrane insertion mechanisms were also analyzed. The targeting of VAPA to the INM was confirmed employing cell culture-based techniques and both confocal and STED microscopy. Its proximity mapping at the ER and INM was determined using RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC). Proximity ligation assays and STED microscopy validated the proximity of the identified proteins. The interaction between VAPA and specific proximity hits was analyzed using co-immunoprecipitation assays, revealing LAP2, emerin, and Nup98 as interaction partners of VAPA. Additionally, we identified a correlation between the depletion or ablation of VAPA with reduced nuclear size, lower expression levels of lamins and nuclear envelope aberrations. In vitro insertion assays using either rough microsomes or semi-permeabilized cells were applied to expand our understanding of the mechanisms involved in the membrane integration of VAPA. Post-translational insertion via the TRC40 pathway was confirmed, although usage of alternative mechanisms was inferred from these results. Collectively, our findings demonstrate the targeting of VAPA to the INM and hint at a potential nuclear role of this protein, such as maintaining the integrity of the lamins and/or the nuclear envelope. Moreover, it strengthens the notion of TA proteins employing alternative membrane integration mechanisms in addition to their canonical insertion pathway.
Keywords: VAPA; Tail-anchored proteins; ER; Nucleus; Nuclear lamina; Inner nuclear membrane proteins; Interactome
