2.A.50 The Glycerol Uptake (GUP) or Membrane-bound Acyl Transferase (MBOAT) Family
Yeast can use glycerol both as a carbon source and as an osmolyte. In Saccharomyces cerevisiae, glycerol has been reported to be actively taken up via two electrogenic H+ symporters, GUP1 (YGL084c) and GUP2 (YPL189w). These proteins are the first characterized members in a moderately sized family of polytopic integral membrane proteins from yeast, fungi, animals and bacteria. Several of the bacterial proteins have been implicated in extracellular polysaccharide modification. One such protein, DltB (spP39580) has been implicated in the transport of activated D-alanine across the bacterial cytoplasmic membrane for biosynthesis of D-alanine lipoteichoic acid. It is possible that proteins implicated in polysaccharide modification serve a similar role. Most of these proteins are of between 450 and 610 aas in length and exhibit 8-10 putative TMSs. One protein (spO09758) from Schizosaccharomyces pombe is half sized, having only 231aas with 4-5 putative TMSs exhibit 8-12 putative TMSs. These proteins are members of the membrane-bound O acyl transferase (MBOAT) family.
The Bacillus subtilis dlt operon (D-alanyl-lipoteichoic acid) is responsible for D-alanine esterification of both lipoteichoic acid (LTA) and wall teichoic acid (WTA). The dlt operon contains five genes, dltA-dltE. Insertional inactivation of dltA-dltD results in complete absence of D-alanine from both LTA and WTA. Based on protein sequence similarity with the Lactobacillus casei dlt gene products (Heaton and Neuhaus, 1992), dltA may encode the D-alanine-D-alanyl carrier protein ligase (Dcl) and dltC the D-alanyl carrier protein (Dcp). The products of dltB and dltD are concerned with the transport of activated D-alanine through the membrane and the final incorporation of D-alanine into LTA. The hydropathy profiles of the dltB and dltD gene products suggest a transmembrane location for the former and an amino-terminal signal peptide for the latter (Perego et al., 1995).
Gup1 homologues of S. cerevisiae and Trypanosoma brucci are glycosyl phosphatidylinositol (GPI) remodelases (Jaquenoud et al., 2008; Ghugtyal et al., 2007). Lipids of Trypanosoma brucei undergo lipid remodelling, whereby longer fatty acids on the glycerol are replaced by myristate (C14:0). A similar process occurs on GPI proteins of Saccharomyces cerevisiae where Per1p first deacylates and Gup1p subsequently reacylates the anchor lipid, thus replacing a shorter fatty acid by C26:0 (Ghugtyal et al., 2007). Heterologous expression of the GUP1 homologue of T. brucei in gup1Delta yeast cells partially normalizes the gup1Delta phenotype and restores the transfer of labelled fatty acids from Coenzyme A to lyso-GPI proteins. Gup1p from T. brucei (tbGup1p) strongly prefers C14:0 and C12:0 over C16:0 and C18:0, whereas yeast Gup1p strongly prefers C16:0 and C18:0. This acyl specificity of tbGup1p closely matches the reported specificity of the reacylation of free lyso-GPI lipids in microsomes of T. brucei. Depletion of tbGup1p in trypanosomes by RNAi drastically reduces the rate of myristate incorporation into the sn-2 position of lyso-GPI lipids. Thus, tbGup1p is involved in the addition of myristate to sn-2 during GPI remodelling in T. brucei and can account for the fatty acid specificity of this process. tbGup1p can act on GPI proteins as well as on GPI lipids. These results and others put into doubt the original claim that GUP1 and GUP2 are glycerol transporters (Bleve et al., 2005; Bosson et al., 2006; Ferreira et al., 2006).
Mammalian glycerol uptake/transporter 1 (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membrane-bound O-acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product. Although mammalian Gup1 has a sequence conserved among the membrane-bound O-acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine. Abe et al. (2007) cloned Gup1 cDNA from adult mouse lung and examined whether it is involved in the regulation of N-terminal palmitoylation of Sonic hedgehog (Shh). Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn. Gup1 and Skn co-localized with endoplasmic reticulum markers, suggesting that these two molecules interact with overlapped targets. Ectopic expression of Gup1 with full-length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity. On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34. Gup1 may thus be a negative regulator of N-terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh (Abe et al., 2007).
Membrane-bound O-acyltransferases (MBOATs) are a superfamily of integral transmembrane enzymes that are found in all kingdoms of life. In bacteria, MBOATs modify protective cell-surface polymers while in vertebrates, some MBOAT enzymes-such as acyl-coenzyme A:cholesterol acyltransferase and diacylglycerol acyltransferase 1-are responsible for lipid biosynthesis or phospholipid remodelling. Other MBOATs, including porcupine, hedgehog acyltransferase and ghrelin acyltransferase, catalyse essential lipid modifications of secreted proteins such as Wnt, hedgehog and ghrelin, respectively. Many MBOAT proteins are important drug targets. Ma et al. 2018 presented crystal structures of DltB, an MBOAT responsible for the D-alanylation of cell-wall teichoic acid, both alone and in complex with the D-alanyl donor protein DltC. DltB contains a ring of 11 peripheral transmembrane helices, which shield a highly conserved extracellular structural funnel extending into the middle of the lipid bilayer. The conserved catalytic histidine residue is located at the bottom of this funnel and is connected to the intracellular DltC through a narrow tunnel. Mutation of either the catalytic histidine or the DltC-binding site of DltB abolishes the D-alanylation of lipoteichoic acid and sensitizes B. subtilis to cell-wall stress, which suggests cross-membrane catalysis involving the tunnel. Structure-guided sequence comparisons among DltB and vertebrate MBOATs reveal a conserved structural core and suggests that MBOATs from different organisms have similar catalytic mechanisms.
The transport reaction proposed to be catalyzed by GUP1 is:
Glycerol (out) + H+ (out) → glycerol (in) + H+ (in)
The transport reaction proposed to be catalyzed by DltB is:
DctC-alanine (in) + Teichoic acid (out) → Alanyl-teichoic acid (out) + DctC (in)