A Novel Sit4 Phosphatase Complex Is Involved in the Response to Ceramide Stress in Yeast

Abstract

Ceramide is a building block for complex sphingolipids in the plasma membrane, but it also plays a significant role in secondary signalling pathways regulating cell proliferation and apoptosis in response to stress. Ceramide activated protein phosphatase activity has been previously observed in association with the Sit4 protein phosphatase. Here we find that sit4Δ mutants have decreased ceramide levels and display resistance to exogenous ceramides and phytosphingosine. Mutants lacking SIT4 or KTI12 display a shift towards nonhydroxylated forms of long chain bases and sphingolipids, suggesting regulation of hydroxylase (SUR2) or ceramide synthase by Sit4p. We have identified novel subunits of the Sit4 complex and have also shown that known Sit4 regulatory subunits—SAP proteins—are not involved in the ceramide response. This is the first observation of separation of function between Sit4 and SAP proteins. We also find that the Sit4p target Elongator is not involved in the ceramide response but that cells deficient in Kti12p—an accessory protein with an undefined regulatory role—have similar ceramide phenotypes to sit4Δ mutants. Therefore, Kti12p may play a similar secondary role in the ceramide response. This evidence points to a novel Sit4-dependent regulatory mechanism in response to ceramide stress.

A personal journey with bioactive lipids

“…We also defined a role for ceramide in activating yeast PP2A , and Nicols and Broach identified the components of the trimer as Sit4p, Cdc55p and Tpd3p . Subsequent studies further corroborated the role of yeast PP2A in mediating actions of ceramide .…”

Sphingolipids and mitochondrial function in budding yeast

“…Sit4p is inactivated by the target of rapamycin complex 1 (TORC1) [56], which is part of the highly conserved target of rapamycin (TOR) nutrient signaling pathway [57]. A regulatory role for Sit4p in SL biosynthesis was recently suggested as Δsit4 mutants display an aberrant SL composition [43]. More specifically, Sit4p was suggested to regulate Sur2p or Cer synthase activity [43].…”

“…A regulatory role for Sit4p in SL biosynthesis was recently suggested as Δsit4 mutants display an aberrant SL composition [43]. More specifically, Sit4p was suggested to regulate Sur2p or Cer synthase activity [43]. Sur2p converts DHS to PHS [58].…”

“…Thus, Sit4p dependent Sur2p or Cer synthase activity [43] could point to a compensatory mechanism for the lack of phytoCer production in Δisc1 mutants. This compensatory mechanism could, for instance, be responsible for the increased generation of αOH-C 14 -phytoCer and C 26 -phytoCer levels in Δisc1 mutant mitochondria [27].…”

“…Likewise, ρ 0 cells are insensitive to Suloctidil and dihydromotuporamine C [33], both compounds that are known to affect SL biosynthesis in mammalian cells [34, 35]. In addition, whereas sub-lethal LCB doses restore viability of yeast mutants defective in SL biosynthesis [36-38] and affect gene expression [39], exogenously added LCBs can kill several fungal species [39-43]. In S. cerevisiae however, loss of the mtDNA increases tolerance to LCBs [44], which is dependent on the retrograde response [44].…”