CFAP43 was predicted to consist of two independently folding domains. The N-terminal domain includes seven WD-repeats, which form a barrel structure, and three coiled-coil stretches are located within the C-terminus (Mai, 2012). Thus, both domains provide plat-forms to bind other proteins raising the possibility that CFAP43 serves as a scaffold protein.
To get insight about the pathways, in which CFAP43 might be acting, putative interaction partners were identified and validated.
2.3.1 Identification of potential interaction partners
Ten putative interaction partners were identified in yeast-two-hybrid screens (Mai, 2012).
These proteins, namely RAD23B, ZBTB32, UBQLN1, BSPRY, MTVR2, ATXN7L1, GRIP1, PDCD7, SFR1 and SNX6, fulfill various tasks in different pathways and cellular processes, including DNA repair, splicing, protein turnover or trafficing, all processes, which are appar-ently unrelated with cilia function. However, some proteins with functions well described in other contexts, such as proteins needed for DNA repair or nuclear trafficking (Attanasio, 2015; Dishinger et al., 2010; Kee et al., 2012), were recently related to cilia function. There-fore, the potential interactions identified by the yeast-two-hybrid screen were investigated by co-immunoprecipitation.
Additionally other potential binding partners, which could possibly be linked to ciliary functions, were identified in cooperation with Dr. Karsten Boldt (Institute for Ophthalmic Research, Universitiy of Tübingen). Therefore plasmids were constructed, which allowed expression of either N-terminally or C-terminally TAP-tagged CFAP43. After successful test-expression of the fusion proteins, CFAP43 was purified by Dr. Karsten Boldt from transiently overexpressing HEK293T cells by tandem-affinity purification using mild washing conditions for co-purification of binding partners. Affinity purified protein complexes were analyzed by mass spectrometry to identify proteins, which were co-purified with CFAP43. A list of 59 proteins was the result of this analysis, which were restricted after grouping of the pro-teins. Six of them are involved in translation and 16 in protein turnover. These proteins were excluded since they could interact with CFAP43 during protein synthesis and turnover and their pull-down might be the consequence of CFAP43 overexpression. Additionally each six nuclear and mitochondrial proteins were ruled out, because no evidence pointed towards a localization of CFAP43 in these organelles. From the remaining 25 proteins two appeared particularly interesting in the frame of this project. The first, IQCB1, was chosen, because it was described to be mutated in some cases of Senior-Løken syndrome (Otto et al., 2005), a well described ciliopathy. The second, CDC42EP1, is associated with the actin cytoskeleton
2.3 Interaction partners
(Liu et al., 2014), which in term is needed for multiciliation of epithelial cells in the airways (Gomperts et al., 2004; Huang et al., 2003; Pan et al., 2007). When analyzing potential in-teraction partners, antibody evaluation as described above was not yet completed. Hence, the potential localization of CFAP43 close to actin remodeling sites could not be excluded at this time. CFAP43 was hypothesized to take part in actin-dependent processes due to the localization visualized by the antibody P4. Therefore, the potential interaction between CFAP43 and CDC42EP1 was evaluated by co-immunoprecipitation. In addition the local-ization of EZRIN or EBP50 in mTECs resembles the signal detected by the antibody P4 (see figure 2.9 A; Gomperts et al., 2004; Huang et al., 2003; Mai, 2012; Pan et al., 2007). Thus, also a putative interaction of CFAP43 with these proteins was investigated, although they were not identified in the screens. 14-3-3 proteins were tested, because CFAP43 was identified in a mass spectrometric analysis subsequent to tandem-affinity purification of 14-3-3ǫ(Zuo et al., 2010).
2.3.2 Validation of selected potential interactors by co-immunoprecipitation
Genes, which have been identified by the yeast-two-hybrid screen were already present in expression plasmids, whereas other genes were isolated from the FANTOM book (Kawai et al., 2001), and coding sequences were used to generate expression vectors coding for C-terminally Flag-tagged proteins. Vectors used for expression of fusion proteins are sum-marized in table 2.2. Expression of genes from these vectors was validated by transfection of CHO cells with the vectors. Except forAtx7l1all genes could be expressed. Subsequently the remaining expression vectors were used together with a plasmid carrying HA-tagged CFAP43 for transfection of CHO cells, and co-immunoprecipitation was performed from transfected cells (figure 2.12). 14-3-3η could not be shown to interact with CFAP43 and is depicted as an example for a co-immunoprecipitation in which no interaction was detected.
Immunoprecipitation of the other proteins are only depicted if interaction (marked by aster-isks) could be shown at least in one direction. Thus, CFAP43 was shown to interact with some, but not all 14-3-3 proteins, as shown by the interactions with 14-3-3βand ǫ, but not η. Interactions with BSPRY and EBP50 were proven both by co-precipitation of CFP43 with BSPRY or EBP50 and vice versa. Interaction with MTVR2, CDC42EP1 and IQCB1 could only be shown by precipitation of the according interaction partner, no co-purification of these proteins could by shown after precipitation of CFAP43. leere zeile
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Table 2.2:Expression plasmids used for co-immunopecipitations
Gene + tag Identification Source Plasmid backbone
Wdr96-HA Mai (2012) pcDNA6.2-DEST
Ywhae-Myc-Flag literature research ORF from Fantom book pCMV6 Ywhab-Myc-Flag similarity toYwhae ORF from Fantom book pCMV6 Ywhah-Myc-Flag similarity toYwhae ORF from Fantom book pCMV6 Ezrin-Myc-Flag literature research ORF from Fantom book pCMV6 Ebp50-Myc-Flag literature research ORF from Fantom book pCMV6
Atxn7l1-Myc-Flag yeast-two-hybrid Origene pCMV6
Cdc42ep1-Myc-Flag mass spectrometry ORF from Fantom book pCMV6 Iqcb1-Myc-Flag mass spectrometry ORF from Fantom book pCMV6
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2.3 Interaction partners
Figure 2.12: CoIP of CFAP43 with potential interaction partners. Some putative interaction partners were tested for physical interaction by CoIP. Each the upper panel shows the western blot probed with anti-HA antibodies, detecting CFAP43-HA, whereas the lower panel depicts the western blot probed with anti-FLAG antibodies, detecting the FLAG-tagged putative interaction partners. Bright green asterisks mark weak interactions, dark asterisks more robust ones. The panel showing 14-3-3η is an example for no detectable interaction.
2.3.3 Further analysis of IQCB1
From the proteins, which were identified as potential binding partners of CFAP43 by the dif-ferent screens and positively validated by co-immunoprecipitation, only IQCB1 was already described to have a ciliary function. Briefly summarized, IQCB1 localizes to the transition zone where it regulates the entry of the BBSome into the cilium. Thereby IQCB1 acts as a gatekeeper for the entry of ciliary proteins (Barbelanne et al., 2015). Before performing a more detailed analysis of this interaction, expression ofCfap43andIqcb1was determined to identify common expression domains and immunoprecipitation was repeated using addi-tional antibodies (figure 2.13). Iqcb1 transcript was detected in all tissues containing motile cilia. Expression of Cfap43was shown in the same tissues (see also figure 2.23 for the com-plete result of this experiment). Additionally both RNAs were detected in human RPE1 cells.
After ensuring coexpression ofCfap43 and Iqcb1 in the same tissues and RPE1 cells, co-immunoprecipitation was repeated using different antibodies for the pull-down of CFAP43.
Figure 2.13: Further validation of a potential interaction between CFAP43 and IQCB1. AThe upper panel depicts expression ofIqcb1in various murine tissues containing motile cilia as shown by RT-PCR using three different primer pairs. For comparison expression ofCfap43in the same tissues is shown in the lower panel (see also figure 2.23). B RT-PCR using each three different primer pairs detect expression ofIqcb1and Cfap43human RPE1 cells. CCoIP with different antibodies as compared to figure 2.12 revealed weak interaction in both tested directions.
Using antibodies P4 or M73, which are directed against CFAP43 instead of the tag, IQCB1 could be co-purified in low amounts. Co-immunoprecipitation by pull-down of IQCB1 worked out as before. Thus, although the bands showing co-purification are rather weak, physical (direct or indirect) interaction between CFAP43 and IQCB1 could be proven. The faint bands of the corresponding co-purified proteins could be explained by transient or weak interactions, as well as indirect interactions, which are mediated by forming complexes with other binding partners being endogenously expressed in CHO cells.
In two screens for identification of putative CFAP43 interaction partners several proteins were identified, which could potentially bind to CFAP43. Some of this potential interac-tions were validated by co-immunoprecipitation. So far, only one of those proteins, IQCB1, has been associated with ciliogenesis. Additionally CFAP43 and IQCB1 were shown to be expressed in the same tissues.