Analysis of the Interactome and Membrane Insertion of VAPB, a Tail- Anchored Protein at the Inner Nuclear Membrane

by Christina James
Doctoral thesis
Date of Examination:2020-06-10
Date of issue:2020-06-26
Advisor:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Stefan Jakobs
Referee:Dr. Ricarda Richter-Dennerlein
Referee:Prof. Dr. Henning Urlaub
Referee:Prof. Dr. Michael Thumm
Referee:Dr. Sven Pd Thoms
Persistent Address: http://dx.doi.org/10.53846/goediss-8061

 

 

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Abstract

English

Tail-anchored (TA) proteins are a group of integral membrane proteins defined by the presence of a single transmembrane domain (TMD) at the C-terminal domain and are involved in functionally diverse cellular processes. Since the C-terminal TMD of TA proteins emerges from the ribosome tunnel only after termination of translation, insertion of these proteins to target membrane occurs mostly by post-translational pathways. Two model TA proteins used in this study are VAPB and emerin.  VAPB (vesicle-associated membrane protein-associated protein B) is an integral endoplasmic reticulum (ER) protein that is present at several contact sites of the ER. To understand the mechanism of insertion of VAPB into the ER, in vitro insertion assays were performed using rough microsomes and semi-permeabilized cells. VAPB was shown to be post-translationally inserted into the ER membrane independently of the TRC40 pathway. Apart from its ER-localization, immunoelectron microscopy and a rapamycin-based dimerization assay showed that VAPB also localizes to the inner nuclear membrane (INM). The engineered ascorbate peroxidase (APEX2) has been effectively employed in mammalian cells to identify protein-protein interactions. Using a modified APEX2-approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, proteins that are in close proximity to VAPB were identified in the ER and the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), followed by co-immunoprecipitation assays, many well-known interaction partners of VAPB at the ER were confirmed and also novel proximity partners at the INM were identified. Hence, rapamycin-APEX2‐mediated proximity labeling of VAPB neighboring proteins provide insights into the VAPB interactome at the ER and the INM.  Emerin is one of the best-characterized tail-anchored proteins of the INM but also localizes to the ER and the outer nuclear membrane (ONM). To better understand the dynamics of emerin at the nuclear envelope (NE), FRAP assays were performed at the NE on intact and permeabilized cells. The addition of cytosol to the permeabilized cells increased the diffusion of emerin to the NE and addition of a Ran deficient mutant, RanQ69L, a lectin wheat germ agglutinin (WGA) and a dominant-negative fragment of Importinß (Impß (45-462)) impaired the diffusion of emerin from the ER to the NE. These data suggest that diffusion of emerin to the NE is dependent on soluble components and thus may underscore a role of soluble factors in diffusion and retention mechanism for targeting of INM proteins. 
Keywords: VAPB; Inner nuclear membrane; Tail anchored proteins; SILAC; Emerin; Dynamics
 
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