A Simple Enrichment Procedure Improves Detection of Membrane Proteins by Immunoblotting

Karginov, Azamat V.; Agaphonov, Michael O.

doi:10.2144/000114474

Cited by 12 publications

(9 citation statements)

References 8 publications

(9 reference statements)

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“…To corroborate the observations that AmtA and its derivatives are localized in the plasma membrane, we carried out a subcellular isolation of plasma membrane from strains grown in 0.1% proline, followed by western blotting using antibodies against GFP (Figure 3B). It is pertinent to mention that membrane fraction was treated with SDS and 8M urea and incubated at room temperature instead of heating at 100°C before subjecting it to SDS electrophoresis, to avoid protein aggregation [73]. Plasma membrane ATPase 1 (Pma1) protein was used as a loading control.…”

Section: Resultsmentioning

confidence: 99%

Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae

Densi

Iyer

Bhat

2022

Preprint

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Role of Horizontal Gene Transfer (HGT) in evolution transcends across the three domains of life. Ammonium transporters are present in all species and therefore offer an excellent paradigm to study protein evolution following HGT. While investigating HGT through complementation assay, we observed that synonymous and nonsynonymous mutations follow an epistastic relationship. As a proxy for HGT, we attempted to complement a mep1mep2mep3Δ strain of S. cerevisiae (triple deletion strain) which cannot grow on ammonium as a sole nitrogen source below a concentration of 3 mM, with amtA of D. discoideum. As the wild type amtA did not complement, we isolated two mutant derivatives of amtA that complemented the triple deletion strain of S. cerevisiae. amtA M1 bears three nonsynonymous and two synonymous substitutions and these substitutions are necessary for its functionality. amtA M2 bears two nonsynonymous and one synonymous substitution, all of which are necessary for functionality. These mutants were then studied at phenotypic, cell biological, and biochemical level. Interestingly, AmtA M1 transports ammonium but does not confer toxicity to methylamine while AmtA M2 transports ammonium as well as confers methylamine toxicity, demonstrating functional diversification. Based on the results presented, we suggest that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions separately. This is because, protein evolution entails an interaction between synonymous and nonsynonymous substitution, which seems to have gone unnoticed thus far. Above observations have significant implications in various facets of biological processes and are discussed in detail.

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Section: Resultsmentioning

confidence: 99%

Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae

Densi

Iyer

Bhat

2022

Preprint

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“…The NMY51 yeast strain was transformed with the prey constructs and grown overnight at 30°C under shaking in 10 ml SD-W medium. Membrane proteins were extracted as described ( Karginov and Agaphonov, 2016 ). The samples were loaded onto a 12% SDS polyacrylamide gel and after separation transferred to Hybond electrochemiluminescence (ECL) nitrocellulose membrane (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).…”

Section: Methodsmentioning

confidence: 99%

Mutational Analysis of the GXXXG/A Motifs in the Human Na+/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function

Palatini

Müller

Lowjaga

et al. 2021

Front. Mol. Biosci.

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Homodimerization is essential for plasma membrane sorting of the liver bile acid transporter NTCP and its function as Hepatitis B/D Virus (HBV/HDV) receptor. However, the protein domains involved in NTCP dimerization are unknown. NTCP bears two potential GXXXG/A dimerization motifs in its transmembrane domains (TMDs) 2 and 7. The present study aimed to analyze the role of these GXXXG/A motifs for the sorting, function, and dimerization of NTCP. The NTCP mutants G60LXXXA64L (TMD2), G233LXXXG237L (TMD7) and a double mutant were generated and analyzed for their interaction with wild-type NTCP using a membrane-based yeast-two hybrid system (MYTH) and co-immunoprecipitation (co-IP). In the MYTH system, the TMD2 and TMD7 mutants showed significantly lower interaction with the wild-type NTCP. In transfected HEK293 cells, membrane expression and bile acid transport activity were slightly reduced for the TMD2 mutant but were completely abolished for the TMD7 and the TMD2/7 mutants, while co-IP experiments still showed intact protein-protein interactions. Susceptibility for in vitro HBV infection in transfected HepG2 cells was reduced to 50% for the TMD2 mutant, while the TMD7 mutant was not susceptible for HBV infection at all. We conclude that the GXXXG/A motifs in TMD2 and even more pronounced in TMD7 are important for proper folding and sorting of NTCP, and so indirectly affect glycosylation, homodimerization, and bile acid transport of NTCP, as well as its HBV/HDV receptor function.

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“…Cultures were harvested during mid‐exponential phase (OD600 = 2) by centrifugation at 3,000 × g for 5 min. Yeast membrane protein extraction was performed according to the protocol of Karginov and Agaphonov (2016). Proteins were resolved on 12% SDS–PAGE and transferred to PVDF membrane.…”

Section: Methodsmentioning

confidence: 99%

Identification, isolation, and heterologous expression of Sunflower wax synthase for the synthesis of tailored wax esters

Shalini

Martin

2020

J. Food Biochem.

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Wax esters (WE) are neutral lipids formed by condensation of fatty alcohol with fatty acyl-CoA by wax synthases. They serve as carbon and energy reserves and are potential substrates for various commercial applications. Sunflower (Helianthus annuus) an edible oil seed is a source of WE, however, the gene responsible for WE formation has hitherto remained unidentified. Using an in silico approach we identified, isolated putative Sunflower wax synthase (HaWS) gene and investigated it's potential for WE production in yeast. Heterologous expression of HaWS in Saccharomyces cerevisiae H1246 exhibited 57 kDa protein which was confirmed by immunoblotting. Recombinant yeast expressing HaWS were fed with combinations of C16, C18 fatty alcohols with 16:0, 18:0 fatty acyl CoA's as potential substrates to validate WE formation in vivo. The yeast cells accumulated C-32 to C-36 WE. Our study reveals identification, isolation, and heterologous functional expression of WS gene from Sunflower for the first time. Practical applications Wax synthases (WSs) are critical enzymes for wax ester (WE) biosynthesis. WEs are high value products having several industrial applications. WE serve as substrates for lubricants, food coatings, cosmetics, and pharmaceuticals. There is a demand for alternate renewable resource of WEs. In this study, we have successfully isolated a putative wax synthase gene from Sunflower and submitted its sequence data to the GenBank (Accession number MH460820). Conserved sequence search analysis showed presence of condensation superfamily motif-HHXXXDG, critical for WE biosynthesis. Heterologous expression of HaWS in yeast revealed synthesis of C-32 to C-36 WE. Our study demonstrates the efficacy of HaWS to accumulate specific WE of desired lengths in yeast, and thus represents an alternate source of WE for commercial applications and for biotechnological production of tailored WE in eukaryotic expression systems.

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A Simple Enrichment Procedure Improves Detection of Membrane Proteins by Immunoblotting

Cited by 12 publications

References 8 publications

Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae

Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae

Mutational Analysis of the GXXXG/A Motifs in the Human Na+/Taurocholate Co-Transporting Polypeptide NTCP on Its Bile Acid Transport Function and Hepatitis B/D Virus Receptor Function

Identification, isolation, and heterologous expression of Sunflower wax synthase for the synthesis of tailored wax esters

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