"Add-on" domains of Drosophila β1,4-N-acetylgalactosaminyltransferase B in the stem region and its pilot protein

Abstract

The glycolipid specific Drosophila melanogaster β1,4-N-acetylgalactosaminyltransferase B (β4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with β4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3-4 and 5-6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, β4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, β4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with β4GalNAcTB, indicating their importance in direct GABPI-β4GalNAcTB interaction.

Genome-wide CRISPR screens for Shiga toxins and ricin reveal Golgi proteins critical for glycosylation

“…We propose that LAPTM4A may act as an activator specifically for A4GALT, and that its second luminal domain facilitates recognition of the glycolipid substrates by A4GALT. A similar case is that of Drosophila β -1,4- N -acetylgalactosaminyltransferase B, which is responsible for synthesis of glycolipids and requires a six-pass membrane protein GABPI for its activity [ 56 , 57 ]. Furthermore, the two short luminal domains of GABPI (12 and 35 amino acids long) are required for the activity of this GT.…”

Identification of a Novel Protein Binding Motif within the T-synthase for the Molecular Chaperone Cosmc

“…Finally, the short but specific stem region of ␤4GalNAcTB is required for its binding to GABPI (65,66), a polytopic ER protein that may act as a type of pilot protein for targeting of ␤4GalNAcTB to the Golgi apparatus. Thus, expanding knowledge of the stem region functions of glycosyltransferases indicate they are important to enzyme function and localization.…”