Transmembrane protein 147 (TMEM147) is a novel component of the Nicalin-NOMO protein complex

Abstract

Nicastrin and its relative Nicalin (Nicastrin-like protein) are both members of larger protein complexes, namely gamma-secretase and the Nicalin-NOMO (Nodal modulator) complex. The gamma-secretase complex, which contains Presenilin, APH-1, and PEN-2 in addition to Nicastrin, catalyzes the proteolytic cleavage of the transmembrane domain of various proteins including the beta-amyloid precursor protein and Notch. Nicalin and its binding partner NOMO form a complex that was shown to modulate Nodal signaling in developing zebrafish embryos. Because its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular masses of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinity-purified complex preparations for additional components in the low molecular mass range. A approximately 22-kDa protein was isolated and identified by mass spectrometry as transmembrane protein 147 (TMEM147), a novel, highly conserved membrane protein with a putative topology similar to APH-1. Like Nicalin and NOMO, it localizes to the endoplasmic reticulum and is expressed during early zebrafish development. Overexpression and knockdown experiments in cultured cells demonstrate a close relationship between the three proteins and suggest that they are components of the same complex. We present evidence that, similar to gamma-secretase, its assembly is hierarchical starting with the formation of a Nicalin-NOMO intermediate. Nicalin appears to represent the limiting factor regulating the assembly rate by stabilizing the other two components. We conclude that TMEM147 is a novel core component of the Nicalin-NOMO complex, further emphasizing its similarity with gamma-secretase.

FIGURE 1.

TMEM147 is a major interactor of Nicalin and NOMO. A, Nicalin-Myc (Ncl-Myc) was immunoprecipitated (IP) from large scale membrane preparations (detergent: 0.7% DDM), and the bound proteins were visualized by Coomassie Blue staining (left panel). In addition to NOMO (∼130 kDa), an ∼22-kDa band, identified as T147, was specifically enriched in precipitates from HEK293T/Ncl-Myc cells (bands denoted with * represent immunoglobulin heavy and light chains). Immunoblotting using a monoclonal anti-TMEM147 antibody confirmed its identity (right panel). B, immunoprecipitation of V5-tagged TMEM147 from overexpressing cells resulted in the specific enrichment of an ∼60- and ∼130-kDa band (left panel) identified as Nicalin and NOMO by immunoblotting (right panel). C, comparison of input, bound (IP), and unbound (post IP) material of Nicalin-Myc (left panel) and TMEM147-V5 (right panel) immunoprecipitations by immunoblotting. A strong enrichment of the respective binding partners (middle lanes) as well as their depletion in the post-immunoprecipitation supernatants (right lanes) is observed. No binding or depletion of the membrane protein calnexin is detectable. e, endogenous.

FIGURE 2.

TMEM147 colocalizes with Nicalin in the ER. A, density gradient centrifugation using membrane protein lysates from HEK293T cells. Collected fractions were analyzed by immunoblotting. The distribution of TMEM147, Nicalin, and NOMO is highly similar with an exclusive presence in the four bottom fractions, which are enriched in ER membranes, as shown by the presence of the ER marker calnexin and of immature (i) Nicastrin. m, mature. B, immunofluorescence microscopy of wild-type HeLa cells and cells overexpressing V5-tagged TMEM147. Nicalin colocalizes with calnexin (upper panel) confirming the ER localization of endogenous Nicalin previously reported (11). Staining for TMEM147 in overexpressing cells resulted in a reticular pattern that largely overlapped with the Nicalin staining, demonstrating colocalization within the ER. (Scale bars, 10 μm.)

FIGURE 3.

TMEM147, Nicalin, and NOMO protein levels are mutually dependent. A, HeLa cells were transduced with lentiviruses encoding the respective short hairpin RNAs to generate stable Nicalin, NOMO, and TMEM147 knockdown (KD) cell lines. Protein levels were analyzed by immunoblotting. The knockdown of Nicalin, NOMO, or TMEM147 resulted in a strong reduction of the respective binding partners. Stable reintroduction of an RNAi-resistant TMEM147 cDNA containing a V5 tag (T147 rescue) restored NOMO and Nicalin levels. wt, wild type. B, mRNA levels in HeLa knockdown cells were quantitated by real-time PCR showing a decrease in target gene expression by 75–90%. The mRNA levels of the interaction partners were only marginally changed. Shown are mean values from three experiments; error bars indicate standard deviations. C, knockdown (KD) and rescue of NOMO and Nicalin in HEK293T cells. TMEM147 expression is reduced in Nicalin and NOMO knockdown cell lines and restored in the rescue cell lines. In a high expression Nicalin rescue clone (Ncl resc9), NOMO and TMEM147 expression are dramatically increased. D, HEK293T cell lines stably overexpressing Nicalin, NOMO, and TMEM147. NOMO overexpression has no effect on Nicalin and TMEM147 levels, whereas Nicalin overexpression results in an increase in NOMO and TMEM147 levels. TMEM147 overexpression does not alter NOMO and Nicalin levels but results in the disappearance of endogenous TMEM147 (replacement). Even the simultaneous overexpression of TMEM147 and NOMO does not lead to an increase in Nicalin expression. Controls are calnexin (left panel) and β-actin (right panel). E, evidence for excess synthesis of TMEM147 and NOMO in HEK293T cells. Nicalin, NOMO, and TMEM147 immunoreactivities were not significantly changed upon ribosome inhibition (chx., cycloheximide, 6 h), indicating a long half-life of the proteins under steady-state conditions. Proteasome inhibition (epox., epoxomicin, 6 h) resulted in an increase in NOMO and TMEM147 levels, indicating the synthesis of excessive protein. F, kinetics of TMEM147 and NOMO stabilization and complex formation in cells with inducible Nicalin expression (293TR/Ncl). Induction of Nicalin expression by tetracycline for the indicated times led to a continuous increase in TMEM147 and NOMO levels over time in lysates (four left lanes) and Nicalin immunoprecipitates (four right lanes), demonstrating enhanced complex formation. The increase of NOMO appears to precede the elevation of TMEM147, an indication for a stepwise complex assembly.

FIGURE 4.

TMEM147 and NOMO bind to different Nicalin domains. A, Nonidet P-40 (NP-40) partially disrupts the complex. Membrane protein lysates containing 1% Nonidet P-40 as detergent were generated from 293T/TMEM147-V5 cells. In TMEM147 immunoprecipitates (IP) NOMO and Nicalin could not be detected (left lane), whereas in Nicalin immunoprecipitates TMEM147 was not found (right lane). B, a schematic illustration of the Nicalin deletion mutants analyzed. C, the Nicalin transmembrane domain is essential for TMEM147 but dispensable for NOMO binding. Nicalin-Myc and the deletion mutants NicalinΔC-Myc and NicalinΔTMC were stably expressed in HEK293T cells, and their effects on NOMO and TMEM147 levels were analyzed (in comparison with wild-type (wt) and NOMO-expressing cells). Whereas NicalinΔC was able to elevate both NOMO and TMEM147, although with a lower efficiency than Nicalin-Myc, in NicalinΔTMC-expressing cells, only NOMO was increased. D, RNAi-insensitive Nicalin-Myc, NicalinΔC, and NicalinΔTMC constructs were stably expressed in HEK293T/Nicalin knockdown (KD) cells. The ability of the Nicalin variants to restore NOMO and TMEM147 expression was analyzed by immunoblotting (when compared with wild-type and Nicalin knockdown cells). NicalinΔC was able to rescue NOMO and TMEM147 expression but was less efficient in elevating it beyond wild-type levels than Nicalin-Myc. In contrast, NicalinΔTMC restored NOMO to wild-type levels but was unable to restore TMEM147 expression.

FIGURE 5.

The interaction between Nicalin, NOMO, and TMEM147 is evolutionary conserved. A, the percentage of amino acid (aa) identity (based on FASTA alignment) and lengths (in amino acids) of Nicalin, NOMO, and TMEM147 orthologs in selected multicellular organisms are shown. H.sapiens, Homo sapiens; M.musculus, Mus musculus; X. laevis, Xenopus laevis; D. rerio, Danio rerio; D.melanogaster, Drosophila melanogaster; A.thaliana, Arabidopsis thaliana. B, effect of zebrafish Nicalin (zfNcl) on the expression of the human orthologs. zfNicalin was stably expressed in HEK293T cells and resulted in the disappearance of endogenous Ncl and in the elevation of endogenous NOMO and TMEM147 levels. Note that zfNicalin migrates slightly higher than human Nicalin and that the amount of overexpressed zfNicalin is underestimated due to a lower affinity of the antibody. wt, wild type. C, zebrafish TMEM147 (zfT147) interacts with human complex partners. Immunoprecipitation of Myc-tagged zfTMEM147 from overexpressing cells resulted in the specific enrichment of endogenous human Nicalin and NOMO. D, zebrafish TMEM147 is expressed during embryonic development. In situ hybridization revealed maternal expression (four-cell stage) and ubiquitous zygotic expression of tmem147 mRNA in fixed zebrafish embryos. The lack of a labeling with the sense probe demonstrated the specificity of the antisense probe. hpf, hours post fertilization.