The serine/threonine protein phosphatase Sit4p activates multidrug resistance in Saccharomyces cerevisiae

Abstract

Multidrug resistance in Saccharomyces cerevisiae is frequently associated with gain-of-function mutations in zinc finger-containing transcription factors Pdr1p and Pdr3p. These regulatory proteins activate the expression of several ATP-binding cassette transporter genes, leading to elevated drug resistance. Here, we report that loss of the type 2A-related serine/threonine protein phosphatase Sit4p renders yeast cells sensitive to cycloheximide, azoles, daunorubicin and rhodamine 6G. This effect is a consequence of the decreased transcriptional levels of mainly PDR3 and its target genes, PDR5, SNQ2 and YOR1, which encode multidrug efflux pumps. The multidrug sensitivity of sit4 mutant cells is suppressed by the PDR1-3 mutant allele, which encodes a hyperactive form of Pdr1p. Sit4p is known to associate with regulatory proteins Sap155p, Sap4p, Sap185p and Sap190p. We found that the sap155 mutant strain is sensitive to azoles, but not to cycloheximide, while the sap155sap4 and sap185sap190 mutant strains are sensitive to both drugs. This finding indicates that the Sit4p-Sap protein complex subtly modulates the expression of drug efflux pumps. Drug resistance conferred by the expression of the Candida albicans CDR1 gene, an ortholog of PDR5 in S. cerevisiae, is also positively modulated by Sit4p. These data uncover a new regulatory pathway that connects multidrug resistance to Sit4p function.

Sphingolipids and mitochondrial function in budding yeast

“…Among these targets are several genes involved in SL biosynthesis such as IPT1, LCB2, SUR2 and LAC1 [31, 32]. In addition, Sit4p affects Pdr3p expression and SL biosynthesis [91]. Whether this effect is mediated via a Pdr3p mechanism is yet to be shown.…”

“…In addition, both Pdr1p and Pdr3p appear important players in multidrug resistance as gain of function mutations in PDR1 and PDR3 increase expression of their target genes encoding multidrug efflux pumps [85]. Furthermore, PDR3 expression increases upon loss of the mitochondrial genome during the retrograde response and is regulated by both Pdr3p itself [86] and Sit4p [91]. …”

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeastSaccharomyces cerevisiae

“…Total protein lysates were prepared as in Miranda et al ., ;. Cells were grown in YPD at 30 °C to the indicated cell densities and harvested by centrifugation at 800 g , for 10 min at 4 °C.…”

“…Disruption of the entire coding region of SIT4 was performed in a BY4741 background using one-step gene targeting with pFa6a-KAN-MX6 (Longtine et al, 1998). The oligonucleotides used are as previously described (Miranda et al, 2010). For the double deletion Dpdc5 Dsit4, a BY4741 Dsit4 background was used by exchanging the KAN (geneticin) marker used in the SIT4 deletion for a NAT (nourseothricin) marker (Hentges et al, 2005).…”

PP2A-Like Protein Phosphatase (Sit4) Regulatory Subunits, Sap155 and Sap190, Regulate Candida albicans’ Cell Growth, Morphogenesis, and Virulence

“…Four regulatory subunits of Sit4 has been identified, and they are named Sit4 association proteins (SAPs) and divided into two groups based on sequence similarity, the SAP4/SAP155 group and the SAP185/SAP190 group (Luke et al, 1996). Studies have shown that the SAPs have diverse functions, such as the regulation of cell growth, K + efflux, and drug resistance (Luke et al, 1996;Manlandro et al, 2005;Miranda et al, 2010). In C. albicans, Sit4 has been identified as the catalytic subunit of PP2A-like protein phosphatase, and deletion of SIT4 causes a significant reduction in growth rate, morphogenesis, and virulence in mice (Lee et al, 2004;Noble et al, 2010).…”