The Cytoplasmic Tail of GM3 Synthase Defines Its Subcellular Localization, Stability, and In Vivo Activity

Abstract

GM3 synthase (SAT-I) is the primary glycosyltransferase responsible for the biosynthesis of ganglio-series gangliosides. In this study, we identify three isoforms of mouse SAT-I proteins, named M1-SAT-I, M2-SAT-I, and M3-SAT-I, which possess distinct lengths in their NH 2 -terminal cytoplasmic tails. These isoforms are produced by leaky scanning from mRNA variants of mSAT-Ia and mSAT-Ib. M2-SAT-I and M3-SAT-I were found to be localized in the Golgi apparatus, as expected, whereas M1-SAT-I was exclusively found in the endoplasmic reticulum (ER). Specific multiple arginines (R) arranged in an R-based motif, RRXXXXR necessary for ER targeting, were found in the cytoplasmic tail of M1-SAT-I, and in vivo GM3 biosynthesis by M1-SAT-I was very low because of restricted transport to the Golgi apparatus. In addition, M1-SAT-I and M3-SAT-I had a long half-life relative to M2-SAT-I. This is the first report demonstrating the presence of an ER-targeting R-based motif in the cytoplasmic tail of a protein in the mammalian glycosyltransferase family of enzymes. The system, which produces SAT-I isoforms having distinct characteristics, is likely to be of critical importance for the regulation of GM3 biosynthesis under various pathological and physiological conditions.

Localization of Golgi-resident glycosyltransferases

“…Amino acid substitution analysis revealed that arginine (R) residues within the CT of Mnn9p, conforming to a canonical R-based COPI-binding motif [66,78], were required for retrieval. Similarly, a recent study by Uemura et al [74] identified an R-based motif in the CT of GM3 synthase that was required for the robust re-cycling of this enzyme from the Golgi to the ER.…”

“…However, the CTs of glycosyltransferases lack canonical COPI-binding motifs so how can these proteins be selected for packaging into COPI coated vesicles? Several recent studies have identified motifs in the CTs of glycosyltransferases that appear to be important for maintaining their steady state distributions [66,74,75], and proteins that bind to the CTs [75][76][77].…”

“…4). Depending on the context, the same set of basic amino acids can function to promote glycosyltransferase export from the ER and retrieval of these enzymes from the Golgi [13,14,66,74,78].…”

Transmembrane BAX Inhibitor Motif Containing (TMBIM) Family Proteins Perturbs a trans-Golgi Network Enzyme, Gb3 Synthase, and Reduces Gb3 Biosynthesis

“…encoded the enzyme with the longest cytoplasmic region among several transcript variants (24). GM3 synthase-expressing cells (rGM3S) showed complete resistance to Stx1 even at higher concentrations (Fig.…”

Quantitative Transcriptomic Profiling of Branching in a Glycosphingolipid Biosynthetic Pathway

“…3, A-D). GM3Syn may occur in alternatively spliced forms (34). Because RT-PCR analysis showed that both Namalwa and Ramos cells expressed the longer isoform of GM3Syn, we used the cDNA of this isoform for further examinations.…”