Detergent-Induced Stabilization and Improved 3D Map of the Human Heteromeric Amino Acid Transporter 4F2hc-LAT2

Meury, Marcel; Costa, Meritxell; Harder, Daniel; Stauffer, Mirko; Jeckelmann, Jean-Marc; Brühlmann, Béla; Rosell, Albert; İlgü, Hüseyin; Kovar, Karin; Palacı́n, Manuel; Fotiadis, Dimitrios

doi:10.1371/journal.pone.0109882

Cited by 25 publications

(39 citation statements)

References 35 publications

(55 reference statements)

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“…Whilst the number of structures obtained using these novel detergents is still much lower than with conventional detergents, this may just be a matter of time. There are reports in the literature of previously challenging proteins that are now able to be extracted and purified to the degree needed for structural studies using these new molecules [45][46][47]. It is interesting to note that both the GNG/MNGs and Calixarenes contain larger, more structured head groups than conventional detergents, and this may be a key factor in their stabilising properties.…”

Section: Discussionmentioning

confidence: 99%

Overcoming bottlenecks in the membrane protein structural biology pipeline

Hardy

Bill

Jawhari³

et al. 2016

Biochemical Society Transactions

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Correspondence to: Alice Rothnie (+44 121 204 4013 a.rothnie@aston.ac.uk) or Anass Jawhari (+33 649 555 606 ajawhari@calixar.com) Key words:Membrane proteins Structural biology SMALP Calixarenes MNG Solubilisation Abbreviations:EM-Electron Microscopy GPCR-G protein-coupled receptors GNG-Glucose Neopentyl Glycol MSP -Membrane Scaffold Protein MNG-Maltose Neopentyl Glycol NMR-Nuclear magnetic resonance SMA-Styrene Maleic Acid SMALPs -SMA Lipid Particles AbstractMembrane proteins account for a third of the eukaryotic proteome, but are greatly underrepresented in the Protein Data Bank. Unfortunately, recent technological advances in X-ray crystallography and electron microscopy cannot account for the poor solubility and stability of membrane protein samples. A limitation of conventional detergent-based methods is that detergent molecules destabilize membrane proteins, leading to their aggregation. The use of orthologues, mutants and fusion tags has helped improve protein stability, but at the expense of not working with the sequence of interest. Novel detergents such as GNG, MNG and calixarene-based detergents can improve protein stability without compromising their solubilising properties. SMALPs focus on retaining the native lipid bilayer of a membrane protein during purification and biophysical analysis.Overcoming bottlenecks in the membrane protein structural biology pipeline, primarily by maintaining protein stability, will facilitate the elucidation of many more membrane protein structures in the near future.

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Section: Discussionmentioning

confidence: 99%

Overcoming bottlenecks in the membrane protein structural biology pipeline

Hardy

Bill

Jawhari³

et al. 2016

Biochemical Society Transactions

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“…To support laborious and time-consuming physiological characterisation of cells based on sophisticated process control, software-sensors have been established (Brühlmann et al, 2014, Herwig et al, 2001, Jenzsch et al, 2006, Luttmann et al, 2012. Such softwaresensors deliver information on the important real-time variables that characterise a bioprocess (such as concentrations of substrates, products and biomass) and are typically determined off-line, with an expected post-sampling delay of several hours.…”

Section: Approaches To Reduce Experimental Loadmentioning

confidence: 99%

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Looser

Brühlmann

Bumbak

et al. 2015

Biotechnology Advances

276

259

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Pichia pastoris, a methylotrophic yeast, is an established system for the production of heterologous proteins, particularly biopharmaceuticals and industrial enzymes. To maximise and optimise the production of recombinant products, recent molecular research has focused on numerous issues including the design of expression vectors, optimisation of gene copy number, co-expression of secretory proteins such as chaperones, engineering of glycosylation and secretory pathways, etc. However, the physiological effects of different cultivation strategies are often difficult to separate from the molecular effects of the gene construct (e.g., cellular stress through over-expression or incorrect post-translational processing). Hence, overall system optimisation is difficult, even though it is urgently required in order to describe and understand the behaviour of new molecular constructs. This review focuses on particular aspects of recombinant protein production related to variations in biomass growth and their implications for strain design and screening, as well as on the concept of rational comparisons between cultivation systems for the development of specific production processes in bioreactors. The relationship between specific formation rates of secreted recombinant proteins, qp, and specific growth rates, μ, has been analysed in a conceptual attempt to compare different systems, particularly those based on AOX1/methanol and GAP/glucose, and this has now evolved into a pivotal concept for bioprocess engineering of P. pastoris.

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“…The system is particularly useful for the expression of membrane proteins for structural studies [ 18 , 19 ]. After an overexpression screening in P. pastoris including nine different heavy and light chains, the human HAT 4F2hc-LAT2 was identified as a promising target for functional and structural studies [ 5 , 20 , 21 , 22 ]. Overexpression followed by affinity purification yielded a functional, recombinant N-terminal His- (4F2hc) and Strep-tagged (LAT2) heterodimeric complex 4F2hc-LAT2 correctly connected by the conserved disulfide bridge [ 5 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

“…After an overexpression screening in P. pastoris including nine different heavy and light chains, the human HAT 4F2hc-LAT2 was identified as a promising target for functional and structural studies [ 5 , 20 , 21 , 22 ]. Overexpression followed by affinity purification yielded a functional, recombinant N-terminal His- (4F2hc) and Strep-tagged (LAT2) heterodimeric complex 4F2hc-LAT2 correctly connected by the conserved disulfide bridge [ 5 , 21 , 22 ]. The amino acid transport activity of the recombinant human complex was either shown by L-leucine uptake into proteoliposomes or whole cell uptake experiments using Pichia cells [ 5 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2

Jeckelmann

Fotiadis

2020

IJMS

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Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris, and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed.

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Detergent-Induced Stabilization and Improved 3D Map of the Human Heteromeric Amino Acid Transporter 4F2hc-LAT2

Cited by 25 publications

References 35 publications

Overcoming bottlenecks in the membrane protein structural biology pipeline

Overcoming bottlenecks in the membrane protein structural biology pipeline

Cultivation strategies to enhance productivity of Pichia pastoris: A review

Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2

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