Share this post on:

Re of fat1 elo3 may not be a neighboring effect but an independent genetic interaction, since Fat1 is the only one of 6 yeast acyl-CoA synthases that can activate very long chain FAs and hence may prepare the substrate for Cst26). Negative genetic interactions appearing in SGA have been utilized before to localize and identify a suppressor mutation in the SSD1 locus, which suppresses growth effects of mutations in the Cbk1 kinase signaling pathway [58]. In our E-MAP the query strains were generated by swapping the kanMX marker for the ura3MX marker so that suppressors of the array strains had a good chance to be transferred to the query strains (see S3 Text, Materials and Methods). This can explain why the phenomenon for all 8 arrows of Fig 12 was seen symmetrically in both query x array as well as array x query plates. We indeed found that the distant deletions that generated the concerted negative S scores in certain chromosomal regions all had either reduced viability, reduced competitive PX-478MedChemExpress PX-478 fitness, a Actinomycin IV web sporulation defect, or reduced respiratory capacity and therefore were susceptible to be overgrown by suppressors. It is conceivable that such undeclared mutations may cause some noise also in other E-MAP studies using the strategies we used. For instance, in another E-MAP study [59], 6 of the 18 negative interactions of tda5 were comprised between YOL108c and YOL27c on the left arm of Chr. XV, the same region as pointed by arrow 4 in Fig 12, although none of these negatively interacting deletions were present in our MSP deletion set. Moreover, there are high correlations among functionally unrelated but regionally concentrated genes also in previously published E-MAPs from other groups [60?2].ConclusionWe tried to do a chemogenetic screen in order to identify lipid flippases, the existence of which has been postulated since a long time based on microsomal assays and structural studies showing that certain acyltransferases have their active site in the lumen of the ER. No obvious candidates emerged from this, but, in view of the unusual detergent sensitivity and permeability of the plasma and ER membranes of flc mutants, a flippase activity of Flc proteins remains a definite possibility, which needs to be pursued by trying to reconstitute Flc proteins into large unilamellar vesicles, e.g. by using and adapting the approaches recently established in our lab [63]. LplT is a lyso-PE transporter of the inner membrane of E.coli [64]. Deltablasting (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) shows some highly significant homologies to 13 yeast genes having > 8 identities covering > 90 of lplT sequence (S2F Table). Ten of them were present in our final array set but none of the 10 was involved in any interaction that got severely aggravated (more negative S score) on Cerulenin. While such homologs remain candidates for GPL flippases, several are localized at the plasma membrane and have well defined transporter functions and the genetic interactions of the others make it unlikely that they would be ER lipidPLOS Genetics | DOI:10.1371/journal.pgen.July 27,20 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip Flopflippases (S2F Table). Another interesting flippase candidate would be the TMEM16 channel homologue IST2, which was not present in our deletion library [65]. Our unexpected observation of genetic interactions of certain genes with deletions in an entire chromosomal region may necessitate some additional filteri.Re of fat1 elo3 may not be a neighboring effect but an independent genetic interaction, since Fat1 is the only one of 6 yeast acyl-CoA synthases that can activate very long chain FAs and hence may prepare the substrate for Cst26). Negative genetic interactions appearing in SGA have been utilized before to localize and identify a suppressor mutation in the SSD1 locus, which suppresses growth effects of mutations in the Cbk1 kinase signaling pathway [58]. In our E-MAP the query strains were generated by swapping the kanMX marker for the ura3MX marker so that suppressors of the array strains had a good chance to be transferred to the query strains (see S3 Text, Materials and Methods). This can explain why the phenomenon for all 8 arrows of Fig 12 was seen symmetrically in both query x array as well as array x query plates. We indeed found that the distant deletions that generated the concerted negative S scores in certain chromosomal regions all had either reduced viability, reduced competitive fitness, a sporulation defect, or reduced respiratory capacity and therefore were susceptible to be overgrown by suppressors. It is conceivable that such undeclared mutations may cause some noise also in other E-MAP studies using the strategies we used. For instance, in another E-MAP study [59], 6 of the 18 negative interactions of tda5 were comprised between YOL108c and YOL27c on the left arm of Chr. XV, the same region as pointed by arrow 4 in Fig 12, although none of these negatively interacting deletions were present in our MSP deletion set. Moreover, there are high correlations among functionally unrelated but regionally concentrated genes also in previously published E-MAPs from other groups [60?2].ConclusionWe tried to do a chemogenetic screen in order to identify lipid flippases, the existence of which has been postulated since a long time based on microsomal assays and structural studies showing that certain acyltransferases have their active site in the lumen of the ER. No obvious candidates emerged from this, but, in view of the unusual detergent sensitivity and permeability of the plasma and ER membranes of flc mutants, a flippase activity of Flc proteins remains a definite possibility, which needs to be pursued by trying to reconstitute Flc proteins into large unilamellar vesicles, e.g. by using and adapting the approaches recently established in our lab [63]. LplT is a lyso-PE transporter of the inner membrane of E.coli [64]. Deltablasting (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) shows some highly significant homologies to 13 yeast genes having > 8 identities covering > 90 of lplT sequence (S2F Table). Ten of them were present in our final array set but none of the 10 was involved in any interaction that got severely aggravated (more negative S score) on Cerulenin. While such homologs remain candidates for GPL flippases, several are localized at the plasma membrane and have well defined transporter functions and the genetic interactions of the others make it unlikely that they would be ER lipidPLOS Genetics | DOI:10.1371/journal.pgen.July 27,20 /Yeast E-MAP for Identification of Membrane Transporters Operating Lipid Flip Flopflippases (S2F Table). Another interesting flippase candidate would be the TMEM16 channel homologue IST2, which was not present in our deletion library [65]. Our unexpected observation of genetic interactions of certain genes with deletions in an entire chromosomal region may necessitate some additional filteri.

Share this post on:

Author: Squalene Epoxidase