Study of TMEM165 signal sequence

Details

Supervisors

Morsomme, Pierre ; Velings, Marie Odile

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

TMEM165 is a human transmembrane protein localized on the cis-side of the Golgi apparatus, linked to type II congenital disorder of glycosylation (CDG) due to mutations in its gene. TMEM165, part of the GDT1 protein family, is hypothesized to function as a Ca2+- Mn2+/H+ antiporter, with its depletion associated with glycosylation defects. This study investigates the role of TMEM165 in cation transport activity and its impact on glycosylation. The main focus is on the correct localization of TMEM165 when fused with a fluorescent protein (mCherry) and the role of its N-terminal signal sequence in this process. Initial experiments showed that N-terminally tagged TMEM165 mislocalized, while C-terminally tagged TMEM165 did not. This led to the hypothesis that a putative signal peptide in the N- terminal region, disrupted by the mCherry fusion, might be responsible. Several constructs were designed to test this hypothesis, including versions of TMEM165 with and without its native signal peptide placed upstream mCherry, as well as replacements with signal peptides from ERP44 and BiP. The results indicate that the N- terminal signal peptide of TMEM165 is crucial for its proper localization within the secretory pathway. The study also suggests that the signal peptide is cleaved during translocation, resulting in a mature protein with six transmembrane domains.