TMEM67 Knockout Cell Line (HEK293)

TMEM67 mutations cause Meckel-Gruber syndrome and other related ciliopathies. TMEM67 is involved in both ciliary transition zone assembly, and non-canonical Wnt signaling mediated by its extracellular domain. How TMEM67 performs these two separate functions is not known. We identify a cleavage motif in the extracellular domain of TMEM67 cleaved by the extracellular matrix metalloproteinase ADAMTS9. This cleavage regulates the abundance of two functional forms: a C-terminal portion which localizes to the ciliary transition zone regulating ciliogenesis, and a non-cleaved form which regulates Wnt signaling. By characterizing three TMEM67 ciliopathy patient variants within the cleavage motif utilizing mammalian cell culture and C. elegans, we show the cleavage motif is essential for cilia structure and function, highlighting its clinical significance. We generated a non-cleavable TMEM67 mouse model which develop severe ciliopathies phenocopying Tmem67 mice, but in contrast, transduces normal Wnt signaling, substantiating the existence of two functional forms of TMEM67.

Glycosylation of membrane proteins plays an essential role in diverse biological processes. However, it remains unknown whether this posttranslational modification occurs on ciliary membrane proteins. Herein, by mass spectrometry-based proteomic analysis, we demonstrate that multiple membrane proteins localized in the ciliary transition zone undergo core fucosylation, an N-linked glycosylation specifically catalyzed by fucosyltransferase 8 (FUT8). In-depth analysis reveals that FUT8 interacts with transmembrane protein 67 (TMEM67), a transition zone component closely linked to ciliopathies, and catalyzes its core fucosylation. Functional investigation shows that core fucosylation stabilizes TMEM67 by impeding its degradation via the autophagy pathway, thereby ensuring its proper localization to the transition zone to promote cilium formation. Fut8-deficient mice exhibit ciliary defects in multiple organs, such as the kidney, brain, and trachea. These findings uncover a critical role for TMEM67 core fucosylation in ciliogenesis and have important implications for the pathogenesis of ciliopathies.