VAPB(vesicle-associated membrane protein-associated protein B) is a tailanchored 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 APEX2 (enhanced ascorbate peroxidase 2)-approach with rapamycindependent 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 non-specific hits. Furthermore, we identified emerin, TMEM43 and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.

Membrane contact sites (MCS) are sites of close apposition of two organelles that help in lipid transport and synthesis, calcium homeostasis and several other biological processes. The VAMP-associated proteins (VAPs) VAPA, VAPB, MOSPD2 and the recently described MOSPD1 and MOSPD3 are tether proteins of MCSs that are mainly found at the endoplasmic reticulum (ER). VAPs interact with various proteins with a motif called FFAT (two phenylalanines in an acidic tract), recruiting the associated organelle to the ER. In addition to the conventional FFAT motif, the recently described FFNT (two phenylalanines in a neutral tract) and phospho-FFAT motifs contribute to the interaction with VAPs. In this review, we summarize and compare the recent interactome studies described for VAPs, including in silico and proximity labeling methods. Collectively, the interaction repertoire of VAPs is very diverse and highlights the complexity of interactions mediated by the different FFAT motifs to the VAPs.

Embryonic stem cells have the ability to self-renew or differentiate and these processes are under tight control. We previously reported that the polyamine regulator AMD1 is critical for embryonic stem cell self-renewal. The polyamines putrescine, spermidine, and spermine are essential organic cations that play a role in a wide array of cellular processes. Here, we explore the essential role of the polyamines in the promotion of self-renewal and identify a new stem cell regulator that acts downstream of the polyamines: MINDY1. MINDY1 protein levels are high in embryonic stem cells (ESCs) and are dependent on high polyamine levels. Overexpression of MINDY1 can promote ESC self-renewal in the absence of the usually essential cytokine Leukemia Inhibitory Factor (LIF). MINDY1 protein is prenylated and this modification is required for its ability to promote self-renewal. We go on to show that Mindy1 RNA is targeted for repression by mir-710 during Neural Precursor cell differentiation. Taken together, these data demonstrate that high polyamine levels are required for ESC self-renewal and that they function, in part, through promotion of high MINDY1 levels. STEM CELLS 2018; 00:000-000 SIGNIFICANCE STATEMENTThis article shows that the polyamines, putrescine, spermidine, and spermine are essential for the maintenance of embryonic stem cell self-renewal. Inhibition of polyamine synthesis causes differentiation. This study identifies the MINDY1 protein as being a critical downstream target of the polyamines in driving self-renewal. MINDY1 functions to remove ubiquitin from target proteins this preventing their degradation. This study proposes that MINDY1 is a new critical regulator of stem cell self-renewal that functions to promote the stability of core self-renewal proteins.

Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin-and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.Cells 2020, 9, 605 2 of 18 three proteins containing this characteristic feature (Lap2, emerin and Man1; [14]). Major binding partners of emerin are A-and also B-type lamins [6]. Indeed, the localization of emerin at the nuclear envelope was shown to depend on lamin A [15]. Hence, retention of emerin upon binding to lamins plays an important role in INM-targeting. Besides lamins, a number of emerin interaction partners have been described, for example BAF (barrier-to-autointegration factor (BANF1) [16], nesprin-1α [17] and HDAC3 [18]; for review see [9]).For proteins of the INM, the identification of interacting proteins is particularly challenging, because conditions that are typically used in, for example, co-immunoprecipitation approaches, may not lead to complete solubilization of NE-structures. Genetic approaches like the yeast-two-hybrid method, on the other hand, detect interactions under rather non-physiological conditions. Neither approach addresses the native subcellular localization of a protein of interest. To overcome these problems, several methods have been developed that use proximity-based labeling, typically introducing biotin into unknown proteins, allowing subsequent affinity capture with immobilized streptavidin and analysis by mass-spectrometry (for review see [19]). One approach termed BioID uses a biotin ligase from E. coli for biotinylation of proteins [20]. As an alternative, ascorbate peroxidase (APEX), an enzyme that generates radicals from biotin phenol in the presence of H 2 O 2 was introduced [21]. Modification of proteins occurs within a range of~20 nm of the enzyme, which can be genetically fused to the Nor C-terminus of proteins of interest for probing their direct environment and identifying potential interaction partners [22][23][24][25]. An improved version of APEX, APEX2, is far more active than the original enzyme [26]. Very recently, we devised a method where APEX2 is not directly fused, but instead targeted to a protei...