Use of Chemical Chaperones in the Yeast Saccharomyces cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-Glycoprotein

Figler, Robert A.; Omote, Hiroshi; Nakamoto, Robert K.; Al‐Shawi, Marwan K.

doi:10.1006/abbi.2000.1712

Cited by 86 publications

(83 citation statements)

References 48 publications

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“…5A), at any value of [K ϩ ] o , compared to more than 10-fold that level of Hak1p ( These results were further emphasized by several experiments with Ncpp, the promoter that normally controls expression of the gene encoding Neurospora's plasma membrane proton pump (NcPMA1). Ncpp itself is not sensitive to [K ϩ ], whether intracellular or extracellular, but it is a routine strong promoter (37,38). Western blot analysis of HA-tagged Trk1p and Hak1p, expressed behind Ncpp rather than their native promoters, found Ncpp to increase Trk1p approximately 10-fold in isolated plasma membranes, regardless of the potassium level (Fig.…”

Section: Growth Experimentsmentioning

confidence: 95%

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Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

et al. 2013

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b Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism Neurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene HAK1 to be much more highly regulated than is TRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K ؉ ] and/or the coexpression of HAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K ؉ and by coexpression of TRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ϳ500:1, viz., 10-to 50-fold larger than that in K ؉ -and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K ؉ stress by means of progressively increasing transporter affinity for extracellular K ؉ . The underlying observations are (iv) that K ؉ influx via Trk1p remains nearly constant at ϳ9 mM/h when extracellular K ؉ is progressively depleted below 0.05 mM and (v) that K ؉ influx via Hak1p remains at ϳ3 mM/h when extracellular K ؉ is depleted below 0.1 mM.

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Section: Growth Experimentsmentioning

confidence: 95%

“…The promoter for the essential gene PMA1, which encodes the major proton pump in fungal plasma membranes (34)(35)(36), is useful as a strong, constitutive promoter (37,38). A plasmid bearing the PMA1 promoter was constructed from plasmid pMF272 (originally designed to target the his3 locus [39]) by modification as follows.…”

Section: Methodsmentioning

confidence: 99%

Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

et al. 2013

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“…This strain grew normally in galactose (Gal) but was defective in growth in glucose (Glc) medium (a condition where the expression from GAL1 promoter is shut down) because ScGPI12 is essential for yeast growth (8). This mutant was then transformed with EhPIG-L cloned into YEpHIS, a yeast expression plasmid (14), and YEpHIS alone (as vector control) and checked for the ability of the vector used for transformation to rescue the growth defect of this mutant in the Glc medium (supplemental Table I). Additionally, microsomes were prepared from all of these strains, in the presence of Gal and Glc, and assayed for the levels of de-N-acetylase activity (supplemental Table I).…”

Section: Methodsmentioning

confidence: 99%

N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase from Entamoeba histolytica

Ashraf¹,

Yadav²,

Sreejith

et al. 2011

Journal of Biological Chemistry

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PIG-L/GPI12 proteins are endoplasmic reticulum-resident membrane proteins involved in the second step of glycosylphosphatidylinositol anchor biosynthesis in eukaryotes. We show that the Entamoeba histolytica PIG-L protein is optimally active in the acidic pH range. The enzyme has an intrinsic low level of de-N-acetylase activity in the absence of metal and is significantly stimulated by divalent cations. Metal binding induces a large conformational change in the protein that appears to improve catalytic rates while not altering the affinity of the enzyme for its substrate.

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“…However, since they interact, presumably, only with their target protein they generally effective at a lower concentration. For instance, known chemical chaperones glycerol and trimethylamine-N-oxide (TMAO) were both found to restore activity of a mutant chloride channel in mice, yet effective serum concentrations were lethal in nearly half the mice [29]. Thus, because some chemical chaperones prove toxic in vivo, pharmacological chaperones are suggested to have great potential for human therapy [21].…”

Section: Chemical Chaperone Applicationsmentioning

confidence: 99%

Unfolding the therapeutic potential of chemical chaperones for age-related macular degeneration

Sauer

Patel

Chan

et al. 2008

Expert Review of Ophthalmology

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SUMMARYRecent studies suggest that pathological processes involved in age-related macular degeneration (AMD) might induce endoplasmic reticulum (ER) stress. Growing evidence demonstrates the ability of chemical chaperones to decrease ER stress and ameliorate ER stress-related disease phenotypes, suggesting that the field of chaperone therapy might hold novel treatments for AMD. In this review, we examine the evidence suggesting a role for ER stress in AMD. Furthermore, we discuss the use of chaperone therapy for the treatment of ER stress-associated diseases, including other neurodegenerative diseases and retinopathies. Finally, we examine strategies for identifying potential chaperone compounds and for experimentally demonstrating chaperone activity in in vitro and in vivo models of human disease.

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Use of Chemical Chaperones in the Yeast Saccharomyces cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-Glycoprotein

Cited by 86 publications

References 48 publications

Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase from Entamoeba histolytica

Unfolding the therapeutic potential of chemical chaperones for age-related macular degeneration

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Use of Chemical Chaperones in the Yeast Saccharomyces cerevisiae to Enhance Heterologous Membrane Protein Expression: High-Yield Expression and Purification of Human P-Glycoprotein

Cited by 86 publications

References 48 publications

Coordination of K + Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

Coordination of K + Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase from Entamoeba histolytica

Unfolding the therapeutic potential of chemical chaperones for age-related macular degeneration

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Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

Coordination of K ⁺ Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated