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 (APP) and Notch. Nicalin and its binding partner NOMO form a complex which was shown to modulate Nodal signaling in developing zebrafish embryos. Since its experimentally determined native size (200-220 kDa) could not be satisfyingly explained by the molecular weights of Nicalin (60 kDa) and NOMO (130 kDa), we searched in affinitypurified complex preparations for additional components in the low-molecular-weight range. A 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 γ-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.