Helical charge distribution at the transmembrane-luminal interface determines subcellular localization

Transmembrane protein localization is typically dictated by short cytosolic tail sequences, whereas luminal domain contribution remains less understood. Here, we identify a short luminal juxtamembrane peptide as a key determinant of subcellular localization for Class I α-1,2 mannosidases—ERManI, ManIA, ManIB, and ManIC—members of the glycoside hydrolase 47 family involved in N-glycoprotein processing. We previously showed that ERManI and ManIA localize to specialized quality control vesicles (QCVs); we now find that ManIB also partially localizes to these vesicles, whereas ManIC is predominantly Golgi-localized. ERManI, ManIA, and ManIB share a conserved luminal juxtamembrane distribution of charged residues, which diverges in ManIC. Structural predictions suggest that this region maintains an α-helical conformation, with the charge pattern oriented on one face. Site-directed mutagenesis that disrupted this charge pattern or altered its helical register (via alanine insertions) shifted localization between QCVs and the Golgi. Moreover, grafting this peptide onto an unrelated transmembrane protein, β-1,3-galactosyltransferase, redirected it from the Golgi to QCVs. These findings suggest that a specific three-dimensional charge pattern at the transmembrane-luminal interface serves as a localization signal. Unlike canonical linear motifs, this mechanism relies on the structural arrangement, revealing a previously unrecognized mode of organelle targeting within the secretory pathway.