Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification.

Bruno Bozaquel-Morais,Mónica Montero-Lomelí,Raffael Schaffrath,Roland Klassen,Valentina D’Angelo,Leonie Vogt

doi:10.3390/biom8030049

Bruno Bozaquel-Morais, Mónica Montero-Lomelí + Show 4 more

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https://doi.org/10.3390/biom8030049

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Abstract

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone.

Highlights

A key step in lipogenesis is catalyzed by acetyl-CoA carboxylase, which in budding yeastSaccharomyces cerevisiae is encoded by the essential ACC1 gene [1]
We reasoned that phenotypes caused by mcm5 s2 U deficiency but not those resulting from additional impacts of the sit4 mutation on Snf1 regulation should respond to upregulated tRNA gene dosage
Since the tRNA overexpression results suggest that only part of the phenotypes of sit4 mutants are caused by the defect in tRNA modification we further investigated a possible role of the modification in regulation of soraphen A resistance and lipid droplet (LD) dynamics, which are known to be strongly affected in sit4 mutants [2]

Summary

Introduction

A key step in lipogenesis is catalyzed by acetyl-CoA carboxylase, which in budding yeastSaccharomyces cerevisiae is encoded by the essential ACC1 gene [1]. Acc catalyzes the conversion of acetyl-CoA to malonyl-CoA and its activity is inhibited via phosphorylation by adenosine monophosphate (AMP)-activated protein kinase (AMPK) Snf1 [2,3]. Snf kinase activity is regulated by glucose availability and the nutrient sensing pathway (target of rapamycin, TORC1) [2,4,5]. The latter is thought to involve the Sit4-Sap190 protein phosphatase, which antagonizes Snf kinase activation. Consistent with this, absence of either Sit or Sap190 results in a constitutively phosphorylated and hyperactive Snf kinase which results in downregulation of Acc activity and a low lipid droplet phenotype, while a SNF1 gene deletion causes an opposite outcome [2]. In further support of this mutual relationship, Biomolecules 2018, 8, 49; doi:10.3390/biom8030049 www.mdpi.com/journal/biomolecules

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