A Six-membrane-spanning Topology for Yeast and Arabidopsis Tsc13p, the Enoyl Reductases of the Microsomal Fatty Acid Elongating System

Abstract

The very long chain fatty acids are crucial building blocks of essential lipids, most notably the sphingolipids. These elongated fatty acids are synthesized by a system of enzymes that are organized in a complex within the endoplasmic reticulum membrane. Although several of the components of the elongase complex have recently been identified, little is known about how these proteins are organized within the membrane or about how they interact with one another during fatty acid elongation. In this study the topology of Tsc13p, the enoyl reductase of the elongase system, was investigated. The N and C termini of Tsc13p reside in the cytoplasm, and six putative membranespanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The N-terminal domain including the first membrane-spanning segment contains sufficient information for targeting to the endoplasmic reticulum membrane. Studies of the Arabidopsis Tsc13p protein revealed a similar topology. Highly conserved domains of the Tsc13p proteins that are likely to be important for enzymatic activity lie on the cytosolic face of the endoplasmic reticulum, possibly partially embedded within the membrane.Cytosolic fatty acid synthases catalyze the de novo synthesis of the 16 or 18 carbons containing fatty acids that are further elongated to very long chain fatty acids by a microsomal enzyme system, the elongase. Very long chain fatty acids are essential components of cuticular waxes and seed triacylglycerols in plants and of several classes of lipids, including the sphingolipids. Sphingolipids function in eukaryotic cells as membrane structural components, in cell interactions with surroundings, and as bioactive molecules involved in signaling and cell regulation. Sphingolipids also interact with sterols to form lipid rafts, which are involved in trafficking of plasma membrane proteins, endocytosis, and protein stability at the cell surface (1-3). The endoplasmic reticulum (ER)2 -associated elongase system is composed of four distinct enzymes that sequentially catalyze condensation between a CoA-esterified fatty-acyl substrate and malonyl-CoA, a 3-ketoacyl-CoA reduction, a 3-hydroxyacyl-CoA dehydration, and a final enoyl-CoA reduction to yield a fatty acid that is two carbon units longer than the primer (4). Several of the genes encoding components of the elongase system were first identified in Saccharomyces cerevisiae (5-9) and later in plants and mammals (10 -16). Several studies indicate that the elongase proteins are organized in a complex within the ER (5,7,17). A complete understanding of the molecular mechanism and organization of the elongase complex will require structural analysis, a challenging prospect because of the intrinsic technical difficulties associated with the purification and crystallization of membrane proteins. However, in the absence of high resolution structural data, detailed topology models aid in the design and interpretation of structure-function studies of membrane proteins. Thus, as a...

Infection-Associated Nuclear Degeneration in the Rice Blast Fungus Magnaporthe oryzae Requires Non-Selective Macro-Autophagy

“…There have, however, been no reports of the functions of VAC8 or TSC13 orthologues in any filamentous fungus to date. The M. oryzae MoTsc13p showed 60.1% identity to S. cerevisiae Tsc13p, with six predicted transmembrane domains (Figure 2 C), consistent with the topology of S. cerevisiae Tsc13p [38]. MoTsc13p has conserved amino acids (Figure S1 B), essential for activity of Tsc13p [38] (Genbank JN977614).…”

“…The M. oryzae MoTsc13p showed 60.1% identity to S. cerevisiae Tsc13p, with six predicted transmembrane domains (Figure 2 C), consistent with the topology of S. cerevisiae Tsc13p [38]. MoTsc13p has conserved amino acids (Figure S1 B), essential for activity of Tsc13p [38] (Genbank JN977614). Importantly, we were unable to find a homologue of S. cerevisiae NVJ1 , using either nucleotide or amino acid sequences of NVJ1 , based on BLASTP or TBLASTN analysis, in the M. oryzae genome database, or by immuno-precipitation which we used to identify proteins interacting with MoVac8-GFP (data not shown).…”

Mutation for Nonsyndromic Mental Retardation in the trans-2-Enoyl-CoA Reductase TER Gene Involved in Fatty Acid Elongation Impairs the Enzyme Activity and Stability, Leading to Change in Sphingolipid Profile

“…C, the predicted membrane topology of the TER enzyme with the P182L mutation site (*) is shown. The prediction was made based on the topology of the yeast and Arabidopsis homologs (20).…”

“…Although the membrane topology of the mammalian TER enzyme has not been determined, it is highly likely that this enzyme is an integral membrane protein with six membrane-spanning domains and cytosolic N and C termini (Fig. 1C) as deduced from the membrane topology of the yeast and Arabidopsis TER homologs (20). In this topology model, the Pro-182 residue is positioned in the second luminal loop.…”

Modifying Vegetable Oils for Food and Non-food Purposes

“…With the exception of a recently characterized elongase from the marine parasitic protozoon Perkinus marinus, all of the elongases that are involved in LCPUFA biosynthetic pathways are of the ELO/SUR4 gene family (Venegas-Caleron et al 2007). Of the remaining proteins involved in elongation, putative beta-ketoacyl-CoA reductases Han et al 2002) and enoyl-CoA reductases (Gable et al 2004;Paul et al 2007) have been suggested for yeast and plants. Recently, the PHS1 gene has been suggested to be responsible for the dehydration reaction in yeast (Denic and Weissman 2007).…”