LPIAT1/MBOAT7 contains a catalytic dyad transferring polyunsaturated fatty acids to lysophosphatidylinositol.

Abstract Human membrane bound O-acyltransferase domain-containing 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase 1 (LPIAT1), is an enzyme involved in the acyl-chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 rs641738 variant has been associated with the entire spectrum of fatty liver disease (FLD) and neurodevelopmental disorders, but the exact enzymatic activity and the catalytic site of the protein are still unestablished. Human wild type MBOAT7 and three MBOAT7 mutants missing in the putative catalytic residues (N321A, H356A, N321A + H356A) were produced into Pichia pastoris, and purified using Ni-affinity chromatography. The enzymatic activity of MBOAT7 wild type and mutants was assessed measuring the incorporation of radiolabeled fatty acids into lipid acceptors. MBOAT7 preferentially transferred 20:4 and 20:5 polyunsaturated fatty acids (PUFAs) to lysophosphatidylinositol (LPI). On the contrary, MBOAT7 showed weak enzymatic activity for transferring saturated and unsaturated fatty acids, regardless the lipid substrate. Missense mutations in the putative catalytic residues (N321A, H356A, N321A + H356A) result in a loss of O-acyltransferase activity. Thus, MBOAT7 catalyzes the transfer of PUFAs to lipid acceptors. MBOAT7 shows the highest affinity for LPI, and missense mutations at the MBOAT7 putative catalytic dyad inhibit the O-acyltransferase activity of the protein. Our findings support the hypothesis that the association between the MBOAT7 rs641738 variant and the increased risk of NAFLD is mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling. Taken together, the increased knowledge of the enzymatic activity of MBOAT7 gives insights into the understanding on the basis of FLD

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

<p>Abstract</p> <p>Background</p> <p>Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory <it>Saccharomyces cerevisiae </it>strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.</p> <p>Results</p> <p>Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory.</p> <p>Conclusions</p> <p>The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory <it>S. cerevisiae </it>strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.</p

A short regulatory domain restricts glycerol transport through yeast Fps1p

The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions. In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fpslp, a member of the major intrinsic protein (MIP) family ]of channel proteins. Here we describe a short regulatory domain that restricts glycerol transport through Fpslp. This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance. It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain. Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required. The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species. The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer. Remarkably, this domain has structural similarity to the channel forming loops B and E of Fpslp and other glycerol facilitators. Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype

Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p

Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators

Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris--functional and kinetic asymmetry revealed in proteoliposomes.

Abstract The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni 2 + -chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [ 3 H]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na + . Internal Na + slightly stimulated the antiport. Optimal activity was found at pH 7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (≥ 70%) and by mercurials and methanethiosulfonates (≥ 80%). Heterologous antiport of [ 3 H]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [ 3 H]glutamine/glutamine antiport Km values of 0.097 and 1.8 mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na + on the external side was 32 mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism

Crystal Structure of a Yeast Aquaporin at 1.15 Å Reveals a Novel Gating Mechanism

Atomic-resolution X-ray crystallography, functional analyses, and molecular dynamics simulations suggest a novel mechanism for the regulation of water flux through the yeast Aqy1 water channel

Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris

<p>Abstract</p> <p>Background</p> <p>When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast <it>Pichia pastoris</it>.</p> <p>Results</p> <p>By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation.</p> <p>Conclusions</p> <p>We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant <it>P. pastoris </it>clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of <it>P. pastoris </it>clones with high expression levels of aquaporins and other classes of membrane proteins.</p

Structure, Function and Regulation of Fps1p. A Eukaryotic Solute Efflux Channel

Integral membrane proteins belonging to the Major Intrinsic Protein (MIP) family can be found in all kingdoms of life. MIPs are transporters of water, small neutral solutes and possibly ions, and hence are important for osmoregulation at different levels. The aim of this thesis has been to study the structure-function relationship of an atypical member of this family, Fps1p, which is a glycerol facilitator from the yeast Saccharomyces cerevisiae. Fps1p is located in the plasma membrane and mediates efflux of the compatible solute glycerol in the cell\u27s adaptation to lower external osmolarity. Fps1p does not contain the two highly conserved NPA motifs in its channel forming loops, which might have consequences for the transport function and/or the specificity of this MIP. Furthermore, Fps1p is exceptionally large compared to other MIPs due to long hydrophilic extensions in both termini, and these domains have been shown to be crucial for the regulation of glycerol transport. Fps1p can be characterised in vivo in whole yeast cells, since its function is reflected in cell growth under different osmotic conditions. A short domain, twelve amino acids from the transmembrane domain in each terminus, has been found to be important for proper regulation. Moreover, the N-terminus seems to act as a restriction domain, since higher activity is observed when this domain is deleted. The mechanism is not clear, but Fps1p is probably gated by conformational changes upon shifts in external osmolarity. The Fps1p membrane core has been modelled based on the known structure of the Escherichia coli glycerol facilitator, GlpF, which is its close homologue. Much effort has been invested in production of pure Fps1p for functional and structural studies. Fps1p has been overexpressed in several systems using the hosts S. cerevisiae, Pichia pastoris and E. coli. In this thesis Fps1p has been compared with MIP proteins to be able to understand more about the structure, transport and the specificity as well as with other solute efflux channels in order to understand more about its regulation

Structure, Function and Regulation of Fps1p. A Eukaryotic Solute Efflux Channel

Integral membrane proteins belonging to the Major Intrinsic Protein (MIP) family can be found in all kingdoms of life. MIPs are transporters of water, small neutral solutes and possibly ions, and hence are important for osmoregulation at different levels. The aim of this thesis has been to study the structure-function relationship of an atypical member of this family, Fps1p, which is a glycerol facilitator from the yeast <I>Saccharomyces cerevisiae</I>. Fps1p is located in the plasma membrane and mediates efflux of the compatible solute glycerol in the cell's adaptation to lower external osmolarity. Fps1p does not contain the two highly conserved NPA motifs in its channel forming loops, which might have consequences for the transport function and/or the specificity of this MIP. Furthermore, Fps1p is exceptionally large compared to other MIPs due to long hydrophilic extensions in both termini, and these domains have been shown to be crucial for the regulation of glycerol transport. <p />Fps1p can be characterised <I>in vivo</I> in whole yeast cells, since its function is reflected in cell growth under different osmotic conditions. A short domain, twelve amino acids from the transmembrane domain in each terminus, has been found to be important for proper regulation. Moreover, the N-terminus seems to act as a restriction domain, since higher activity is observed when this domain is deleted. The mechanism is not clear, but Fps1p is probably gated by conformational changes upon shifts in external osmolarity. The Fps1p membrane core has been modelled based on the known structure of the <I>Escherichia coli</I> glycerol facilitator, GlpF, which is its close homologue. Much effort has been invested in production of pure Fps1p for functional and structural studies. Fps1p has been overexpressed in several systems using the hosts <I>S. cerevisiae, Pichia pastoris and E. coli</I>. <p />In this thesis Fps1p has been compared with MIP proteins to be able to understand more about the structure, transport and the specificity as well as with other solute efflux channels in order to understand more about its regulation