The TRK1 potassium transporter is the critical effector for killing of Candida albicans by the cationic protein, Histatin 5.

The principal feature of killing of Candida albicans and other pathogenic fungi by the catonic protein Histatin 5 (Hst 5) is loss of cytoplasmic small molecules and ions, including ATP and K(+), which can be blocked by the anion channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. We constructed C. albicans strains expressing one, two, or three copies of the TRK1 gene in order to investigate possible roles of Trk1p (the organism's principal K(+) transporter) in the actions of Hst 5. All measured parameters (Hst 5 killing, Hst 5-stimulated ATP efflux, normal Trk1p-mediated K(+) ((86)Rb(+)) influx, and Trk1p-mediated chloride conductance) were similarly reduced (5-7-fold) by removal of a single copy of the TRK1 gene from this diploid organism and were fully restored by complementation of the missing allele. A TRK1 overexpression strain of C. albicans, constructed by integrating an additional TRK1 gene into wild-type cells, demonstrated cytoplasmic sequestration of Trk1 protein, along with somewhat diminished toxicity of Hst 5. These results could be produced either by depletion of intracellular free Hst 5 due to sequestered binding, or to cooperativity in Hst 5-protein interactions at the plasma membrane. Furthermore, Trk1p-mediated chloride conductance was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in all of the tested strains, strongly suggesting that the TRK1 protein provides the essential pathway for ATP loss and is the critical effector for Hst 5 toxicity in C. albicans.

Epitope tagging of the yeast K(+) carrier Trk2p demonstrates folding that is consistent with a channel-like structure.

TRK family proteins, which mediate the concentrative uptake of potassium by plant cells, fungi, and bacteria, resemble primitive potassium channels in sequence and have recently been proposed actually to fold like potassium channels in a 4-MPM motif (Durell, S. R., and Guy, H. R. (1999) Biophys. J. 77, 789 - 807), instead of like conventional substrate porters in the 12-TM motif (Gaber, R. F., Styles, C. A., and Fink, G. R. (1988) Mol. Cell. Biol. 8, 2848-2859). The known fungal members of this family possess a very long hydrophilic loop, positioned intracellularly in the K(+)-channel model and extracellularly in the substrate porter model. This and two shorter hydrophilic segments have been tested as topological markers for the true folding pattern of TRK proteins using Saccharomyces cerevisiae Trk2p. Hemagglutinin epitope tags were inserted into all three segments, and the enhanced green fluorescent protein (EGFP) was fused to the C terminus of Trk2p. The gene constructs were expressed from a high copy plasmid, and sidedness of the tags was determined by native fluorescence (EGFP), indirect immunofluorescence, and immunoelectron microscopy. Both the long-loop tag and the C-terminal EGFP fusion allowed abundant protein to reach the plasma membrane and support normal yeast growth. In all determinations, the long-loop tag was localized to the inner surface of the yeast cell plasma membrane, thus strongly supporting the channel-like folding model. Additional observations showed (i). membrane-associated Trk2p to lie in proteolipid rafts; (ii). significant tagged protein, expressed from the plasmid, to be sequestered in cytoplasmic vesicular-tubular clusters; and (iii). suppression of such clusters by yeast growth in 5-10% glycerol. This chaperone-like effect may assist other membrane proteins (overexpressed or heterologously expressed) to function within the yeast plasma membrane.

Expression, Purification and Biological Activity Analysis of Human Vascular Endothelial Growth Factor (VEGF(165)) in Pichia pastoris.

The human VEGF(165) cDNA was amplified by PCR, and was inserted, after confirming by DNA sequence analysis, into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and lead sequence of alpha factor gene to form a recombinant expression plasmids pPIC9K/hVEGF(165). This recombinant plasmid was transformed into KM71. Transformants were screened, cultured inflasks and induced by the addition of 1% methanol. After 4 days of methanol induction, the expressed hVEGF(165) came up to 60% of total proteins in medium supernatant as shown by SDS-PAGE. Western blot assay proved that the expressed hVEGF(165) had good antigenicity and high specificity. The recombinant protein was further purified by using Heparin-Sepharose CL6B affinity chromatography, and was proved that it had good biological activity to stimulate HUVEC proliferation and to promote collateral blood vessel formation in an acquired limb artery occlusion animal model.

Ligand Binding Domains of human Vascular Endothelial Growth Factor Receptor.

Four cDNA clones, encoding truncated Flt-1 mutants consisting of loop 2, 1-2, 2-3 and 1-3, were amplified by PCR from human placenta cDNA library and the corresponding gene fragments were named Flt-1(2) Flt-1(1-2) Flt-1(2-3) and Flt-1(1-3). In order to detect which part of the extracellular domain was involved in ligand binding, the interaction between Flt-1 mutants and hVEGF(165) was studied with yeast two-hybrid system. The data presented here suggest that both Flt-1(2-3) and Flt-1(1-3) were able to bind hVEGF(165). Two recombinant expression plasmids, pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3), were constructed and transformed into Pichia pastoris strain GS115, respectively. After 4 days of methanol induction, the amount of the expressed Flt-1s reached 60% of total proteins in supernatant by SDS-PAGE. Recombinant proteins were purified with CM-Sepharose Fast Flow and Sephacryl S-100 chromatography. Biological activities analysis proved that 1-3 loop had slightly better biological activity than 2-3 loop in VEGF(165) binding and in inhibition of HUVEC proliferation stimulated by hVEGF(165).