The transfer membrane, indicating similar loading on the proteins for every single sample.which include Kir family members channels to complement the development of K + uptakedeficient Saccharomyces cerevisiae strain, e.g., SGY1528 that lacks K + uptake transporters Trk1 and Trk2 [12], in low K + media has proven especially useful for identification of novel trafficking pathways of membrane proteins. For example, Shikano et al. applied SGY1528 cells to screen library of Kir2.1 channels which can be fused with C-terminal random 8-mer peptide sequences and identified a group of cis-acting sequences that specifically interact with 14-3-3 proteins and market surface expression of membrane proteins [13]. Furthermore, the screening of a cDNA library transduced into the Kir2.1 channel-expressing SGY1528 cells resulted in a discovery of a trans-acting protein that enhances the cell surface expression of Kir2.1330765-27-9 site 1 [14]. The SGY1528 has been also effectively utilized to identify the structural determinants for the K + selectivity of G-protein gated inwardly-rectifying K + channel(GIRK) [15]. On the other hand, Kolacna and colleagues have developed K + efflux-deficient S. cerevisiae strain, B31. This strain lacks a Na + ATPase and a Na + /H + antiporter and therefore shows development inhibition in higher K + media as a result of excessive accumulation of intracellular K + upon heterologous expression of a mammalian Kir2.1 channel [16]. Possessing the truth that Kir2.1 activity inversely correlates with all the B31 development in high K + , we explored the possibility that B31 growth assay may be conveniently utilized to systematically screen for the structural determinants of K + channel functions and for the novel signal motifs that down-regulate cell surface expression of membrane proteins by a gain-of-function (i.e., cell survival) assay. As a proof of concept, we examined (1) if distinct mutations that disrupt functions of Kir2.1 is often represented by B31 tolerance to high K + , (2) if otherJoshua D. Bernstein et al. / FEBS Open Bio three (2013) 196?Fig. 2. B31 growth inhibition in high K + media represents KCNK channel activities on cell surface. (A) Alignment of C-terminal sequences from KCNK3 and KCNK9 channels. (B) Association of COPI and 14-3-3 proteins with KCNK3 (upper panels) and KCNK9 (lower panels). HEK293 cells transfected with HA-KCNK3 or Myc-KCNK9 have been lysed and immunoprecipitated with HA or Myc Abs. The eluants were resolved by SDS AGE and immunoblotted for the associating -COP and 14-3-3 at the same time as HA (KCNK3) or Myc (KCNK9). (C) Surface expression of KCNK3 and KCNK9.82954-65-2 Price HEK293 cells have been transfected with HA-tagged rat KCNK3 or Myc-tagged human KCNK9 and analyzed for cell surface expression by FCM.PMID:33602106 For KCNK3 (upper panels), the expression is shown in histograms as well as the Median values were determined for the total cell populations as described for Fig. 1. The histograms of KCNK3-transfected cells (filled) had been overlaid with that of vector-transfected cells (unfilled). For KCNK9 (reduced panels), the expression is shown in density plots (see Section two) exactly where x-axis indicates the fluorescence intensity inside a logarithmic scale and y-axis indicates the side scatter (SSC) on the cell. The Median values have been determined for the cells that were positive for the KCNK9 signal (shown in squares inside the density plots). The bar graphs indicate the Median values in average ?s.d. of triplicate samples in the representative of three unique experiments. (D) Development assay of KCNK-expressing B31. The KCNK- or pYES.