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The Nicalin-NOMO complex has been shown to be involved in the Nodal signaling pathway in zebrafish embryos (Haffner et al., 2004), but the molecular mechanism mediating this effect is unknown. Based on the ER-localization of the complex, two modes of action were conceivable: an influence on the secretion of the ligand Nodal or a regulation of the trafficking of Nodal receptors or co-receptors. However, co-isolation and co-regulation studies using these molecules did not lead to clear results and made an unbiased approach necessary to identify possible interactors which could provide a link between the complex and the Nodal pathway. The approach used to identify NOMO and TMEM147 - Nicalin affinity purification with subsequent protein staining - proved not sensitive enough for the detection of transient interactors. Therefore, the purification was combined with mass spectrometry analysis to identify co-purified proteins. To minimize the detection of false positives, a common problem in mass spectrometry approaches, stable isotope labeling using amino acids in cell culture (SILAC) was used and only hits with a heavy/light ratio above 1.8 were considered true interactors resulting in a list of 36 proteins. To identify the most promising interactors, signal intensity and protein coverage data were included in the analysis in addition to the heavy/light ratio (see Tab. 2). NOMO as well as TMEM147 were among the top hits confirming the suitability of this approach. Its specificity was demonstrated by the fact that nine of the ten top hits were ER-associated proteins. However, none of the potential interactors represented an obvious link to the observed phenotypes from animal studies, i.e. Nodal signaling or nAChR assembly (see sections 1.2 and 3.5). The most interesting group of proteins on the hit list were four SNARE proteins - Syntaxin 18, BNIP1, Sec22b and p31 - previously shown to form the Syntaxin 18 complex involved in the retrieval of proteins from the Golgi apparatus back to the ER (Hatsuzawa et al., 2000). The interaction between Nicalin and all four members of the SNARE complex was confirmed by co-isolation/immunoblotting and co-localization was demonstrated by immunofluorescence microscopy. An effect on the function of this complex by the Nicalin-NOMO complex could result in alterations in the trafficking of molecules involved in Nodal signaling. Alternatively, the Syntaxin 18 complex might also be responsible for the ER retrieval of the Nicalin-NOMO complex. However, SNARE proteins mediate the fusion of vesicles to target membranes, but are usually not involved in the recruitment and binding of cargo proteins in the respective vesicles (reviewed by Wickner and Schekman, 2008). Adapter proteins, on the other hand, that mediate this recruitment, were not among the hits. In addition, the Syntaxin 18 complex has been attributed other important functions such as ER subdomain organization (Iinuma et al., 2009). Thus, the relevance of this interaction will need further investigation.

Another interesting group among the potential Nicalin interactors were ER-residual proteins implicated in protein translocation through the ER membrane, such as Grp78/Bip, Ddost (dolichyl-diphosphooligosaccharide-protein glycosyltransferase) 48 kDa subunit, SRPR (signal-recognition particle receptor) subunit β, SPC (signal-peptidase complex) 22 kDa subunit, SPC (signal-peptidase complex) 18 kDa subunit, the translocon subunits Sec61 α and Sec61 β and ribosomal proteins. These interactions could not be explained by the precipitation of nascent Nicalin interacting with translocation factors, as the precipitated Nicalin was tagged C-terminally. Interestingly, a recent proteomics study (Gilchrist et al., 2006), which analyzed the secretory pathway by the isolation of its compartments and subsequent mass spectrometry, indicated an enrichment of Nicalin and NOMO in the rough ER, where translocation occurs. Moreover, the distribution of Nicalin and NOMO between rough ER, smooth ER and Golgi apparatus observed in that study was very similar to that of the translocon component Sec61 α (Fig. 42).

Following the rationale of the study (Gilchrist et al., 2006), this ‘co-clustering’ is an indication for a function of the Nicalin-NOMO complex in the context of protein translocation into the ER. Indeed, the proteins enriched in the ‘Sec61 α cluster’ comprised translocation factors such as Ddost subunits, translocon subunits and ribosomal proteins. On the other hand, it also contained proteins unrelated to translocation such as enzymes involved in ER lipid metabolism, while proteins involved in translocation such as signal peptidase were found in different clusters. Thus, further (biochemical) data are needed to proof or disproof a functional relationship between Nicalin and the translocation machinery. Yet, further support for an involvement of the Nicalin-NOMO complex in translation/ER quality control came from Fig. 42: ‘Co-clustering’ of proteins of the early secretory pathway. A) Rough ER membrane (RM), smooth ER membrane (SM), and Golgi (G) data (n = 3) were averaged and a heat map was generated. Clusters containing Sec61, p97 and Calnexin as markers were selected (Pearson correlation coefficient > 0.90). B) Mean values for proteins in each of these three clusters were plotted individually (normalized % total peptides). Both Nicalin and NOMO showed a similar RM/SM/G distribution as the translocon subunit Sec61 α, indicating a functional relationship. Adapted from Gilchrist et al., 2006.

a study that analyzed the interactions of the secreted human hepatic lipase (HL) during its maturation in chinese hamster ovary (CHO) cells by tandem-affinity purification (TAP) and mass spectrometry (Doolittle et al., 2009; Fig. 43). 16 interacting ER proteins were identified, including NOMO (but not Nicalin) and components of the two major ER chaperone systems, the BiP/Grp94 and the Calnexin/Calreticulin system. However, NOMO was only found to interact when cells were stressed using dithiothreitol, a reducing agent that causes protein unfolding, indicating that the interaction is only triggered under enhanced stress conditions, which might lead to artificial interactions.

Some of the potential Nicalin interactors were identical to γ-secretase interactors identified recently by a study based on a tandem-affinity purification (TAP)/mass spectrometry approach (Wakabayashi et al., 2009). Among these proteins were voltage dependent ion channel (VDAC), ER lipid raft enriched (Erlin) proteins, sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), dolichyl-diphosphooligosaccharide-protein-glycosyltransferase (Ddost) subunits and Sec22b, indicating that these factors may be common contaminations of (ER) membrane affinity purifications and/or (maybe less likely) they generally interact with (ER) membrane protein complexes.

In summary, the combination of SILAC, co-isolation and mass spectrometry presented in this work is a promising approach for the identification of novel Nicalin interactors. However, subsequent studies will have to analyze the identified interactions in more detail. The described analysis needs to be repeated in wild-type cells with endogenous Nicalin to avoid overexpression artifacts. To that end, an anti-Nicalin antibody has to be generated which allows the precipitation of the intact complex. Alternatively, another purification approach could be developed to purify the endogenous complex, as has been achieved for the γ- secretase complex (Winkler et al., 2009).

Fig. 43: Hepatic lipase may interact with NOMO during maturation. Diagram comparing ER proteins co-purifying with TAP- tagged hepatic lipase (HL-TAP) in untreated CHO cells or cells treated with dithiotreitole (DTT), a reducing agent that causes protein unfolding. The shaded areas represent proteins that are the strongest candidates for roles in HL maturation and/or degradation. (Nodal modulator 1, NOMO1). Adapted fromDoolittle et al., 2009

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