Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Costa, Meritxell; Rosell, Albert; Álvarez-Marimon, Elena; Zorzano, António; Fotiadis, Dimitrios; Palacı́n, Manuel

doi:10.1016/j.pep.2012.10.003

Cited by 24 publications

(55 citation statements)

References 41 publications

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“…To investigate this possibility, we studied the behavior of purified 4F2hc/LAT2 and LAT2. Solubilization with DDM of isolated membranes from yeast cells coexpressing His-4F2hc and Strep-LAT2 and sequential purification by Co 2+ -and Strep-Tactin affinity chromatography yielded 4F2hc/LAT2 heterodimers (18). In contrast, DDM solubilization of yeast membranes expressing Strep-LAT2 alone and subsequent Strep-Tactin affinity chromatography and sizeexclusion chromatography yielded only aggregates of LAT2 (Fig.…”

Section: Significancementioning

confidence: 92%

“…According to this model, 4F2hc-ED interacts with all the extracellular loops or ends of TMDs of LAT2. 4F2hc is N-glycosylated in Pichia (18) and mammalian cells (8), and in the model the four putative sites are located in the most external face of 4F2hc-ED, out of the contact interface with LAT2 (Fig. 2B).…”

Section: Significancementioning

confidence: 94%

“…The indicated tagged versions of human 4F2hc and LAT2 were expressed and purified as reported previously (18). L-leucine transport by Pichia cells was performed as described (18).…”

Section: Methodsmentioning

confidence: 99%

“…L-leucine transport by Pichia cells was performed as described (18). Transport activity of LAT2, 4F2hc, and 4F2hc/ LAT2 was tested as described previously (13) by measuring their L-leucine/ L-isoleucine exchange activity in reconstituted proteoliposomes.…”

Section: Methodsmentioning

confidence: 99%

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Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Rosell

Meury

Álvarez-Marimon

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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116

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Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.CD98hc | 4F2hc ectodomain H eteromeric amino acid transporters (HATs) are composed of two subunits, a heavy (SLC3 family) and a light subunit [SLC7 or L-type amino acid transporter (LAT) family] linked by a conserved disulfide bridge (1). HATs are amino acid exchangers (1), and this transport activity resides in the light subunit (2). The heavy subunit (either 4F2hc or rBAT) is essential for trafficking of the holotransporter to the plasma membrane (3, 4). In mammals, six transporters heterodimerize with 4F2hc, and only one heterodimerizes with rBAT. The rBAT/b 0,+ AT complex is a dimer of heterodimers in which the light subunit is required for proper rBAT folding and stability (5, 6). In contrast, 4F2hc-associated transporters are simple heterodimers (6), and possible stabilizing roles of the two subunits in the biogenesis of the heterodimer have not been described.HATs have major impacts on human health and are involved directly in amino acidurias (cystinuria and lysinuric protein intolerance), tumor cell growth, glioma invasion, Kaposi's sarcomaassociated herpesvirus infection, and cocaine relapse (1). In addition to the role of HATs in amino acid transport, 4F2hc heterodimers mediate β1-and β3-integrin signaling (7).Structural information about HATs is scarce (1). The heavy subunits are type II membrane N-glycoproteins with a single transmembrane domain (TMD), an intracellular N terminus, and a large extracellular C terminus with sequence homology with bacterial α-amylases. Indeed, the atomic structure of the extracellular domain (ED) of human 4F2hc (4F2hc-ED) is similar to that of bacterial glucosidases [i.e., a triose phosphate isomerase barr...

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Section: Significancementioning

confidence: 92%

Section: Significancementioning

confidence: 94%

“…The indicated tagged versions of human 4F2hc and LAT2 were expressed and purified as reported previously (18). L-leucine transport by Pichia cells was performed as described (18).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Rosell

Meury

Álvarez-Marimon

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

116

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“…However, the bottleneck of these studies is represented by the overlapping functions of LATs transporters and of other amino acid transporters, which hamper the measurements of affinity constants as well as the single contribution of each amino acid transporter in pharmacokinetics. Very interestingly, LAT1/CD98 and LAT2/CD98 heterodimers have been heterologously over-expressed in E.coli and P.pastoris, respectively shedding light on important functional and structural aspects (Costa et al, 2013;Galluccio et al, 2013). In particular, the availability of studying such proteins isolated from the cellular context, represents an essential condition for drug interaction analysis.…”

Section: Slc7a5 and Slc7a8: Lat1 And Lat2mentioning

confidence: 99%

Amino Acid Transporters in Drug Discovery

Scalise¹,

Pochini²,

Galluccio³

et al. 2014

Current Research in Drug Discovery

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Administered drugs interact with membrane transporters of epithelia, Blood Brain Barrier and other districts influencing their delivery and efficacy. Drugs can also be used as inhibitors of transporters involved in human pathology. Drug-transporter interactions are responsible of off-target effects contributing to toxicity. High Throughput Screening technologies increased the potential applications in therapy or in predicting side effects. These strategies will be helpful in reducing animal experimentation. The identification of transporters important for drug absorption, delivery and side effect production and the best technologies for studying interactions are the main goals in this field. Amino acid transporters are not yet considered in human therapy in spite of their involvement in several pathologies. The function of the amino acid transporters EAAT1, ASCT2, GLYT2, GLYT1, B0AT1, LAT1 and LAT2 is so far well characterized. Some structural data on these transporters have also been obtained by bioinformatics.Interactions of these proteins with several drugs have been well defined at the molecular level. Large scale and, in some cases, high throughput screening of pharmacological compounds make these transporters of particular interest and potential application in human health.

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Cloning, Large Scale Over-Expression in E. coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter

Galluccio¹,

Pingitore²,

Scalise³

et al. 2013

Protein J

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The high yield expression of the human LAT1 transporter has been obtained for the first time using E. coli. The hLAT1 cDNA was amplified from HEK293 cells and cloned in pH6EX3 vector. The construct pH6EX3-6His-hLAT1 was used to express the 6His-hLAT1 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected 8 h after induction by IPTG at 28 °C. The expressed protein was collected in the insoluble fraction of cell lysate. On SDS-PAGE the apparent molecular mass of the polypeptide was 40 kDa. After solubilization with sarkosyl and denaturation with urea the protein carrying a 6His N-terminal tag was purified by Ni(2+)-chelating affinity chromatography and identified by anti-His antibody. The yield of the over-expressed protein after purification was 3.5 mg/L (cell culture). The human CD98 cDNA amplified from Imagene plasmid was cloned in pGEX-4T1. The construct pGEX-4T1-hCD98 was used to express the GST-hCD98 protein in the Rosetta(DE3)pLysS strain of E. coli. The highest level of expression was detected in this case 4 h after induction by IPTG at 28 °C. The expressed protein was accumulated in the soluble fraction of cell lysate. The molecular mass was determined on the basis of marker proteins on SDS-PAGE; it was about 110 kDa. GST was cleaved from the protein construct by incubation with thrombin for 12 h and the hCD98 was separated by Sephadex G-200 chromatography (size exclusion). hCD98 showed a 62 kDa apparent molecular mass, as determined on the basis of molecular mass markers using SDS-PAGE. The yield of CD98 was 2 mg/L of cell culture.

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Expression of human heteromeric amino acid transporters in the yeast Pichia pastoris

Cited by 24 publications

References 41 publications

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

Amino Acid Transporters in Drug Discovery

Cloning, Large Scale Over-Expression in E. coli and Purification of the Components of the Human LAT 1 (SLC7A5) Amino Acid Transporter

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