Validation of the interaction of the selected binders with Xpo7

So far, we identified a new set of proteins that interacted with Xpo7 in a RanGTP dependent or RanGTP sensitive manner. The latter suggests that Xpo7 might function as an importin, supporting our initial findings with Xpo7 affinity

chromatography. Although some of the RanGTP sensitive binders were cytoplasmic proteins, most of them were nuclear; therefore, the import of these proteins by Xpo7 was an attractive possibility. In order to validate the binding of these candidates to Xpo7, I selected nine candidates and performed pull-downs and binding assays. Moreover, I included a candidate from the potential export cargo list to these assays. A short summary of the selected candidates will follow:

Single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is a glycosylase that removes uracil from preferentially single-stranded DNA and therefore functions in base excision DNA repair (Haushalter et al., 1999; Masaoka et al., 2003). Histone-binding protein RBBP7 (RBBP7) and histone acetyltransferase type B catalytic subunit (HAT1) are the subunits of the type B histone acetyltransferase (HAT-B) complex. HAT-B complex acetylates soluble H4 and is though to play a role in nucleosome assembly (Verreault et al., 1998;

Makowski et al., 2001). Guanosine-3,5-bis(diphosphate) 3-pyrophosphohydrolase MESH1 (MESH1) is a cytoplasmic protein that functions in starvation response (Sun et al., 2010). Copper homeostasis protein cutC homolog (CutC) can bind to Cu¹⁺ and may function in copper homeostasis (Li et al., 2005). Porphobilinogen deaminase (HMBS) is another cytoplasmic protein of the import cargo candidates and is involved in the heme biosynthesis by catalyzing the head to tail condensation of four porphobilinogen molecules into hydroxymethylbilane (UniProt, 2015). Protein O-GlcNAcase (MGEA5) cleaves N-acetylglucosamine from O-glycosylated proteins and can function both in the nucleus and cytoplasm (Gao et al., 2001). Histone deacetylase 8 (HDAC8) is a member of the class I histone deacetylases and plays a role in transcriptional regulation (Buggy et al., 2000; Hu et al., 2000; Van den Wyngaert et al., 2000). Nicotinamide phosphoribosyltransferase (NAMPT) is involved in the NAD biosynthesis and is responsible for the production of nicotinamide mononucleotide (Rongvaux et al., 2002). It might also function as adipokine in the bloodstream (Romacho et al., 2013). Finally, alpha and beta subunits (RABGGTA and RABGGTB) of the type II geranylgeranyl transferase are the only tested export candidates. This enzyme transfers the geranylgeranyl moiety to various Rab proteins and directs them to the corresponding membrane (Farnsworth et al., 1994).

Figure 3-47 Identified candidate proteins enrich Xpo7 from a cytoplasmic extract in a RanGTP regulated manner

0.5 pmol H14-ZZ-Nedd8 tagged candidates were immobilized to Z-affibody dimer beads. Then, the beads were incubated with 250 µL cytoplasmic HeLa extract either supplemented with 5 µM RanGTP or same volume of buffer. After 3 h incubation, unbound proteins were removed; the immobilized cargoes and bound proteins were eluted by incubating the beads with 250 nM bdNEDD8 protease. Both input and eluates were separated by SDS-PAGE. One gel was analyzed by Coomassie staining (upper panel). The other one was analyzed by western blotting with anti–hsXpo7 antibody (lower panel). The area corresponding to the highlighted area in the upper panel is shown in the lower panel. Potential export and import cargoes are colored in green and red, respectively. Red asterisks mark the corresponding cargoes.

I cloned these candidates into a vector containing His14-ZZ-NEDD8 tag. I also cloned RhoGAP into a similar construct to use as a positive control for Xpo7 binding. All of these constructs were expressed in E. coli. RBBP7 and RabGGTA were not soluble; therefore I did not include these proteins in the further either by Coomassie-staining or by western blotting using an anti-humanXpo7

– + – + – + – + – + – + – + – + – + – + – + ^{Input RhoGAP} CutC MESH1 NAMPT HMBS MGEA5 HDAC8 HAT1 SMUG1RABGGTB

antibody (Figure 3-47). All of the tested proteins enriched Xpo7 from the extract, indicating that these proteins in fact interact with Xpo7. RhoGAP and RabGGTB enriched Xpo7 only in the presence of RanGTP and therefore behaved like export cargoes. In contrast, in the other proteins, Xpo7 enrichment significantly reduced in the presence of RanGTP, which was a characteristic of the import substrates. It should be noted that the Xpo7 binding in MESH1, NAMPT and RabGGTB was relatively weaker compared to the other proteins.

Figure 3-48 Binding assay with candidates and recombinant Xpo7

0.5 µM H14-ZZ-NEDD8 tagged cargoes were incubated with 0.75 Xpo4 in the absence or presence of 1.5 µM RanGTP in 500 µL at 50 mM NaCl. After 2 h incubation at 4 °C the samples were incubated with Z-affibody dimer beads to capture the cargoes and bound proteins. After another 2 h incubation, unbound proteins were removed, RanGTP and bound proteins were eluted by incubating the beads with 250 nM bdNEDD8 protease.

Eluates were separated by SDS-PAGE and analyzed by Coomassie staining. Potential export and import cargoes are colored in green and red, respectively. Red asterisks mark the corresponding cargoes. Cargoes on the left and on the right were tested on different dates, as a control RhoGAP was included in both panels.

Similar experiment at 100 mM NaCl gave identical results. CutC^ΔNLS represents the construct where the classical nuclear localization signal (RKRAR) on CutC was mutated to TGSAT to test if the interaction depends on NLS.

We have found that all of the selected candidates interact with Xpo7, however, this interaction could have happened directly or via an adaptor molecule. Therefore I wanted to test the direct interaction with recombinantly expressed Xpo7. I followed the same experimental procedure described above. Instead of a cytoplasmic extract, I used recombinant Xpo7 and tested the Xpo7 binding of these cargoes in the absence and presence of RanGTP. Figure 3-48 shows the SDS-PAGE analysis of the experiment. Xpo7 binding, although very weak for some proteins, was seen in all proteins, indicating that these proteins contain contact sites for

– + – + + – – + – + – + – + – + – + – + – + – + RhoGAP CutC CutC^∆NLSMESH1 NAMPT HMBS MGEA5 HDAC8 RhoGAP HAT1 SMUG1RABGGTB

200

Xpo7. RhoGAP and RabGGTB bound to Xpo7 only in the presence of RanGTP.

Additionally, the potential import cargo HDAC8 also bound to Xpo7 only in the presence of RanGTP, and hence behaved like an export cargo in the isolated context. Moreover, the Xpo7 binding of HMBS, MGEA5 and SMUG1 was also different than the previous experiments. These proteins bound to Xpo7 in a RanGTP independent manner. In other words, RanGTP presence did not affect Xpo7 binding to these proteins in this given experimental setting. Xpo7 binding to CutC and NAMPT was as expected and took place only in the absence of RanGTP. Finally, MESH1 and HAT1 also bound to Xpo7, however, the bound Xpo7 was fairly low.