Importance of KUP8 for K+ uptake in rooted plantlets of Elaeis guineensis under K+ sufficient conditions

Husri, Mohd Naqiuddin; Ong‐Abdullah, Meilina

doi:10.1016/j.sajb.2018.06.010

Cited by 2 publications

(2 citation statements)

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“…Application of the split-GFP system has been demonstrated to reduce the risk of GFP interference (Fucile et al, 2002;Rodriguez-Banqueri et al, 2012). Switching to E. coli system may also be one of the alternatives considering that it was possible to express functional EgKUP in E. coli (Husri and Ong-Abdullah, 2018). Using E. coli as an expression system to characterise KT/KUP/ HAK transporters was also proven successful for the barley HvHAK2 (Senn et al, 2001).…”

Section: Future Perspectivesmentioning

confidence: 99%

“…In plants, a few members of the KT/KUP/HAK transporters have been demonstrated to play a key role in potassium acquisition particularly when K + availability is low (Banuelos, 2002;Rodriguez-Navarro, 2006;Gierth, 2005;Martínez-Cordero et al, 2004;Santa-Maria et al, 1997;Wang, 2002). There are also evidences that members of KT/ KUP/HAK transporters are involved in diverse physiological functions such as complementing the role of K + -channels for low-affinity K + -transport (Fu and Luan, 1998;Rigas et al, 2001;Husri and Ong-Abdullah, 2018), response to salinity (Senn et al, 2001;Garciadeblas et al, 2002;Takahashi et al, 2007) and regulation of cell size (Elumalai et al, 2002;Grabov, 2007). Despite their physiological importance, our understanding on KT/KUP/HAK membrane transporters remains limited due to difficulties in working with this class of proteins.…”

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confidence: 99%

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EVALUATION OF GFP-BASED SYSTEM FOR PRE-CRYSTALLISATION STUDY OF EgKUP MEMBRANE PROTEINS

Husri¹

2019

JOPR

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Elucidation of atomic structures of membrane proteins (MP) is of great importance for comprehensive understanding of their functions and mechanism of actions. Determination of high resolution protein structures by X-ray crystallography requires crystallisation of a protein of interest. Obtaining wellordered protein crystals however, is not an easy task and many factors can contribute to unsuccessful crystallisation. The situation will get much trickier when dealing with MP owing to the fact that they are not soluble in their innate state after extraction from the lipid bilayer environment. Unless appropriate detergents are used to stabilise proteins by mimicking their native environment, MP tend to unravel, thus becoming dysfunctional. This study evaluates the effectiveness of using Green Fluorescence Protein (GFP)-based system as C-terminal tag in Saccharomyces cerevisiae for pre-crystallisation study of Elaeis guineensis MP from the K + Transporter/ K + Uptake Permease/High Affinity K + (KT/KUP/ HAK) superfamily. KT/KUP/HAK are one of the most important MP responsible for potassium uptake in plants. Development of recombinant expression system for high-level protein production is necessary as the vast majority of MP cannot be obtained in sufficient quantities from their native environment. The precrystallisation analyses were conducted on three EgKUP-GFP fusions, namely EgKUP3-GFP, EgKUP8-GFP and EgKUP11-GFP. The analyses involved whole-cell florescent count of each fusion protein using spectrofluorometer, protein integrity assay by SDS-PAGE, cellular localisation by confocal fluorescence microscopy and effects of various detergents towards EgKUPs' stability and monodispersity using Fluorescence Size Exclusion Chromatography (FSEC). Results from the evaluation, associated problems and proposed troubleshootings are discussed.

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Section: Future Perspectivesmentioning

confidence: 99%