Golgi localization of GnTI requires a polar amino acid residue within its transmembrane domain.

The Golgi apparatus consists of stacked cisternae filled with enzymes to facilitate the sequential and highly controlled modification of glycans from proteins that transit through the organelle. Although the glycan processing pathways have been extensively studied, the underlying mechanisms that concentrate Golgi resident glycosyltransferases and glycosidases in distinct Golgi compartments are unclear. The single-pass transmembrane domain of N-acetylglucosaminyltransferase I (GnTI) accounts for its steady-state distribution in the cis/medial-Golgi. Here, we investigated the contribution of individual amino acid residues from the GnTI transmembrane domain for Golgi localization and N-glycan processing. Conserved sequence motifs within the transmembrane domain were replaced with those from the well-known trans-Golgi enzyme alpha2,6-sialyltransferase (ST) and site-directed mutagenesis was used to exchange individual amino acid residues. Subcellular localization of fluorescent fusion proteins and N-glycan profiling revealed that a conserved glutamine residue in the GnTI transmembrane domain is essential for its cis/medial-Golgi localization. Replacing the crucial glutamine residue with other amino acids results in mislocalization to the vacuole and impaired N-glycan processing in vivo. Our results suggest that sequence-specific features in the transmembrane domain of GnTI are required for interaction with a Golgi-resident adaptor protein or specific lipid environment that likely promotes COPI-mediated retrograde transport to maintain the steady-state distribution of GnTI in the cis/medial-Golgi.