Organization of glycolipid biosynthetic enzymes in the Golgi complex

Glycolipids play diverse biological roles — from serving as receptors of toxins and growth factors to overseeing molecular recognition at the cell surface. These molecules are composed of a ceramide backbone to which monosaccharide (sugar) molecules are attached. The order in which different sugars are attached to the ceramide backbone is a crucial feature in determining the precise role of a glycolipid. Understanding the machinery that regulates the glycosylation of the ceramide backbone, therefore, is critical to our understanding of the biosynthesis of these important lipids and provides insight into the mechanisms underlying diseases resulting from aberrations of their synthesis. Interestingly, the organization of the glycosylation machinery within the Golgi is linked intimately to the supramolecular organization and dynamics of the Golgi complex itself.
 
The enzymes responsible for catalyzing the stepwise addition of monosaccharides to ceramide and ceramide-bound oligosaccharides are the glycolipid glycosyltransferases, or GGTs (reviewed in Yu et al). Researchers have given considerable effort to understanding the organization of GGTs within the Golgi.
 

Maccioni-figure
A simplified scheme of ganglioside biosynthesis, showing the topological distribution
of the GGT in Golgi compartments as deduced from the block imposed by
Brefeldin A (marked gray) in metabolic labeling studies with CHO-K1 cells.

Like other Golgi glycosyltransferases, GGTs that use GlcCer and more complex glycolipids as acceptors show a modular organization, with their N-terminal domain bearing a transmembrane domain flanked by a short cytoplasmic tail and a flexible lumenal stem region to which a large C-terminal domain bearing the sugar nucleotide and acceptor glycolipid binding sites is appended (reviewed in Varki et al).

Notably, the GGTs are unlike glycoprotein glycosyltransferases, which alternate with glycosidases in the processing of oligosaccharides, in that they are not strictly distributed along the Golgi in the order in which they act. Instead, they organize as distinct homo- and hetero-multienzyme complexes, which in many cases involve their N-terminal domains.
 
Deducing the sub-Golgi localization and organization of GGTs has been challenging. Our current understanding results mainly from studies involving activity determinations in Golgi subfractions, cell metabolic labeling in the presence of pharmacological reagents that disrupt Golgi dynamics and structure like Brefeldin A, single-cell imaging, and immunoprecipitation of epitope-tagged versions (reviewed in Maccioni et al). Such studies have demonstrated interesting differences in the organization of specific GGTs.
 
For example, in CHO-K1 cells, the N-terminal domains of GalT1, SialT1 and SialT2 (see Fig. 1) participate in a heterocomplex in the Golgi and the trans-Golgi network, while the complex formed by GalNAcT and GalT2 localizes instead in the trans-Golgi network. Recent studies in FAPP2 knockout mice reveal that the synthesis of Gb3 in the TGN uses LacCer synthesized from GlcCer transported by nonvesicular (FAPP2) intermediates, while the track for synthesis of GM3 in the Golgi uses LacCer synthesized from GlcCer transported by vesicular intermediates.
 
GGT organization, however, is only part of the story. How GGTs and Golgi-resident proteins are maintained in the Golgi while plasma-membrane-destined proteins exit the Golgi remains a mystery. Bioinformatics data indicate that transmembrane domains, or TMDs, of Golgi proteins (which represent about 80 percent of putative glycosyltransferases) are four to five amino acids shorter than those of plasma-membrane proteins (see Sharpe et al and Quiroga et al), but the significance of this to GGT organization is unclear.
 
An interaction of amino-acid motifs at GGTs’ cytoplasmic tails with coat proteins has been proposed to mediate retention in yeast. Additionally, it has been suggested that a hydrophobic mismatch between the short TMDs of some glycosyltransferases and the increased bilayer thickness of Golgi membranes at export domains, due to the addition of order-inducing sphingolipids and cholesterol and/or to the concentration of proteins with long TMDs, segregate them from these domains.
 
Studies using chimeric proteins have provided some hints on this point. Swapping the hemi-transmembrane domains of Golgi and plasma-membrane-resident proteins (the yeast SNAREs Sft1 and Sso1, respectively) showed that, in addition to the short length, the presence of voluminous amino acids in the exoplasmic hemi-TMD is a crucial parameter for Golgi localization. Proteins with longer TMDs and less voluminous exoplasmic halves exit the Golgi and localize to the plasma membrane both in yeast and in mammalian cells.
 
These studies highlight the role of the shape of TMDs in the fitness of glycosyltransferases (or their oligomeric associations) in either processive or export lipid domains of Golgi membranes.
 
Clearly, we are learning a lot about the localization and topology of these important enzymes. But the story is far from complete. Studies like these, as well as others intended to enhance our understanding of these enzymes, will keep the glycolipid community busy for a long time.

 

Hugo MaccioniHugo J.F. Maccioni (hugomaccioni@
gmail.com) is a professor emeritus at the National University of Córdoba in Argentina.

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