Abstract
Vesicle-associated membrane protein-associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.
Highlights
Vesicle-associated membrane protein–associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER)
Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, at the inner nuclear membrane (INM)
Using a version of APEX2 that accumulates in the nucleus, we identified additional neighboring proteins of VAPB that reside at the nuclear envelope, e.g. emerin, TMEM43, lamins, and the nucleoporin embryonic large molecule derived from yolk sac (ELYS; AHCTF1)
Summary
Vesicle-associated membrane protein–associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively. Using this method (rapamycin- and APEX-dependent identification of proteins by SILAC or RAPIDS), we found RMDN3 (PTPIP51), ACBD5, YIF1A, OSBPL9, and other previously known interacting proteins of VAPB.
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