The Suppressor of AAC2 Lethality SAL1 Modulates Sensitivity of Heterologously Expressed Artemia ADP/ATP Carrier to Bongkrekate in Yeast

Monika Wysocka-Kapcinska,Lilla Turiak,Roza Kucharczyk,George Tsaprailis,Beata Torocsik,Karoly Vekey,Christos Chinopoulos,Vera Adam-Vizi,Cynthia L David,Andrea M Hunt

doi:10.1371/journal.pone.0074187

Abstract

The ADP/ATP carrier protein (AAC) expressed in Artemia franciscana is refractory to bongkrekate. We generated two strains of Saccharomyces cerevisiae where AAC1 and AAC3 were inactivated and the AAC2 isoform was replaced with Artemia AAC containing a hemagglutinin tag (ArAAC-HA). In one of the strains the suppressor of ΔAAC2 lethality, SAL1, was also inactivated but a plasmid coding for yeast AAC2 was included, because the ArAACΔsal1Δ strain was lethal. In both strains ArAAC-HA was expressed and correctly localized to the mitochondria. Peptide sequencing of ArAAC expressed in Artemia and that expressed in the modified yeasts revealed identical amino acid sequences. The isolated mitochondria from both modified strains developed 85% of the membrane potential attained by mitochondria of control strains, and addition of ADP yielded bongkrekate-sensitive depolarizations implying acquired sensitivity of ArAAC-mediated adenine nucleotide exchange to this poison, independent from SAL1. However, growth of ArAAC-expressing yeasts in glycerol-containing media was arrested by bongkrekate only in the presence of SAL1. We conclude that the mitochondrial environment of yeasts relying on respiratory growth conferred sensitivity of ArAAC to bongkrekate in a SAL1-dependent manner.

Highlights

Embryos of the brine shrimp Artemia franciscana exhibit a type of extremophilia characterized by tolerance of anoxia at room temperature for several years [1] [2] with no evidence of apoptotic or necrotic cell death [3]
As the double aac2 sal1 deletion strain is lethal and a functional Sal1p is required for growth of yeast in the presence of bongkrekic acid (BKA) which blocks the operation of AAC2 protein [20], the Artemia AAC (ArAAC) was expressed in SAL1 and sal1::NatMX4 deletion background
We investigated the effect of heterologously expressing a>] the adenine nucleotide carrier (AAC) of Artemia franciscana in Saccharomyces cerevisiae

Summary

Introduction

Embryos of the brine shrimp Artemia franciscana exhibit a type of extremophilia characterized by tolerance of anoxia at room temperature for several years [1] [2] with no evidence of apoptotic or necrotic cell death [3]. Protracted anoxia in mammalian species opens the so-called mitochondrial permeability transition pore (PTP) [4], [5], [6]. This pore is of a sufficient size (cut-off ,1,5 kDa) to allow passage of solutes and water, causing swelling and rupture of the organelle. Artemia franciscana is the only species known in which adenine nucleotide exchange operated by AAC is refractory to the naturally occurring inhibitor, bongkrekic acid (BKA) [14]. We have reported that mitochondria obtained from brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibit BKA-sensitive mitochondrial adenine nucleotide transport while lacking a Ca2+-induced permeability transition [18]

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Conclusion