A cleavable signal peptide controls the topology and Golgi targeting of the membrane protein TMEM165

TMEM165 is a Golgi-resident multi-pass membrane protein involved in divalent cation homeostasis and associated with congenital disorders of glycosylation, yet its N-terminal biogenesis has remained unresolved. Here, we demonstrate that TMEM165 contains a functional cleavable signal peptide required for correct Golgi targeting and membrane topology. Loss of this signal peptide causes protein mislocalization, and altered topology with N-terminal cytosolic exposure, whereas extended N-terminal deletion restores both Golgi localization and overall membrane topology, consistent with insertion mediated by the first transmembrane domain as commonly described for multi-pass membrane proteins. Importantly, this N-terminally truncated form remains responsive to manganese-induced degradation and partially restores glycosylation defects associated with TMEM165 deficiency, indicating that the extended N-terminal region is dispensable for core TMEM165 function. Together, these findings identify the signal peptide as a key determinant of TMEM165 biogenesis and suggest that its conservation may contribute not only to membrane targeting, but also to maintaining the proper luminal environment of the N-terminus during early biogenesis.