Expression of FLR1 Transporter Requires Phospholipase C and Is Repressed by Mediator

Abstract

In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in benomyl sensitivity, alterations in chromatin structure of centromeres, mitotic delay, and a higher frequency of chromosome loss. Here we intended to utilize benomyl sensitivity as a phenotype that would allow us to identify genes that are important for kinetochore function and are downstream of Plc1p. However, our screen identified SIN4, encoding a component of the Mediator complex of RNA polymerase II. Deletion of SIN4 gene (sin4⌬) does not suppress benomyl sensitivity of plc1⌬ cells by improving the function of kinetochores. Instead, benomyl sensitivity of plc1⌬ cells is caused by a defect in expression of FLR1, and the suppression of benomyl sensitivity in plc1⌬ sin4⌬ cells occurs by derepression of FLR1 transcription. FLR1 encodes a plasma membrane transporter that mediates resistance to benomyl. Several other mutations in the Mediator complex also result in significant derepression of FLR1 and greatly increased resistance to benomyl. Thus, benomyl sensitivity is not a phenotype exclusively associated with mitotic spindle defect. These results demonstrate that in addition to promoter-specific transcription factors that are components of the pleiotropic drug resistance network, expression of the membrane transporters can be regulated by Plc1p, a component of a signal transduction pathway, and by Mediator, a general transcription factor. The results thus suggest another layer of complexity in regulation of pleiotropic drug resistance.The hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C (PLC) 3 yields two prominent eukaryotic second messengers, 1,2-diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ). In higher eukaryotes, the hydrophilic IP 3 triggers the release of calcium from internal stores and thus modulates Ca 2ϩ /calmodulin-regulated pathways, whereas the hydrophobic 1,2-diacylglycerol activates the phospholipid-and Ca 2ϩ -dependent protein kinase C (1-3).In yeast cells, PLC (Plc1p encoded by PLC1) and three inositol polyphosphate (InsPs) kinases (Ipk2p/Arg82p, Ipk1p, and Kcs1p) constitute a nuclear signaling pathway that affects transcriptional control (4) and export of mRNA from the nucleus (5). Recently, InsPs produced by a Plc1p-dependent pathway have been shown to regulate the activity of chromatin remodeling complexes in vivo and in vitro (6, 7). The induction of the phosphate-responsive PHO5 gene, chromatin remodeling of its promoter, and recruitment of Swi/Snf and Ino80 chromatin remodeling complexes are impaired in the ipk2/arg82 mutant strain (7). In vitro, nucleosome mobilization by the yeast Swi/Snf complex is stimulated by IP 4 and IP 5 , whereas IP 6 inhibits nucleosome mobilization by yeast Isw2 and Ino80 complexes and Drosophila NURF complex (6). A possible mechanism by which InsPs affect chromatin remodeling may involve effects on protein conformation of the chromatin remodeling complexes (7). Alternativ...

Inositol polyphosphates: a new frontier for regulating gene expression

“…Future studies will determine whether 6PP-IP 5 , its stereoisomeric cousin 4PP-IP 5 , or both participate in PHO signaling. To date, a number of studies have strengthened the role of IPs in transcriptional control (Auesukaree et al 2005;El Alami et al 2003;Odom et al 2000;Romero et al 2006 Fig. 3 Model for global control of gene expression by IPs in the PHO pathway.…”

Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology

“…For instance, Ipk2 is required for transcriptional responses other than through the ArgR-Mcm1 complex (Auesukaree et al, 2005;El Alami et al, 2003;Odom, 2002Odom, , 2000Romero et al, 2006;Shen et al, 2003;Steger et al, 2003). It is also possible that some of the pleiotropic defects observed occur as a result of metabolic changes downstream of Ipk2, such as altered PP-IP 4 and/or PP-IP 5 .…”

Facilitated Assembly of the Preinitiation Complex by Separated Tail and Head/Middle Modules of the Mediator

“…27 Plc1p is a budding yeast phospholipase C that is required for the initial step of inositol polyphosphates (InsPs) synthesis. InsPs regulate activity of chromatin remodeling complexes in vivo and in vitro 28,29 and are required for induction of the phosphate-responsive PHO5 gene.…”