Catalytic Rate of transporter HXT2

Value 53 glucose molecules/sec
Organism Budding yeast Saccharomyces cerevisiae
Reference Kruckeberg AL, Ye L, Berden JA, van Dam K. Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein. Biochem J. 1999 Apr 15 339 ( Pt 2):299-307. abstract, p. 303 left column, top sentence & p.306 left column bottom paragraphPubMed ID10191260
Method "To investigate further the properties and regulation of Hxt2 researchers have tagged it with the green fluorescent protein (GFP) of Aequorea victoria. GFP matures after translation to be an intrinsically fluorescent protein." "[Investigators] have constructed an Hxt2-GFP fusion protein for use as a reporter of Hxt2 expression, abundance and localization within the yeast cell."
Comments "[Investigators] calculated from the fluorescence level and transport kinetics that induced cells had 1.4x10^5 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30° C." "The cellular abundance of the Hxt2-GFP chimaera and the glucose transport activity mediated by that protein were used to calculate the catalytic-centre activity for glucose of the transporter. The Hxt2-GFP cell suspension contained 0.61 mg of total cell protein/ml, giving a relationship of 32 pmol of Hxt2-GFP/mg of total cell protein. The Vmax for glucose transport of these cells, 103 nmol/min per mg of total cell protein (Table 3) was divided by this value, resulting in an estimate of 53 s^-1 for the catalytic-centre activity of Hxt2-GFP. All of the Hxt2-GFP in the cells seemed to be at the plasma membrane when examined by fluorescence microscopy (results not shown)." "By quantifying the emission from GFP in the fusion protein [investigators] were able to determine a value for the catalytic-centre activity of Hxt2-GFP in vivo. [They] estimate that under inducing conditions each hxt-null cell transformed with single-copy HXT2-GFP has 1.4×10^5 Hxt2 molecules in the plasma membrane and that the transporter has a catalytic-centre activity at 30 °C of 53 s^-1. To [their] knowledge this is the first empirical estimate of a catalytic centre activity for a yeast hexose transport protein. It assumes that all Hxt2-GFP molecules were actively transporting glucose and is therefore a minimum estimate." See BNID 101737, 101738
Entered by Ben Marks
ID 101739