Functional expression of human adenine nucleotide translocase 4 in Saccharomyces cerevisiae

Abstract

The adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP across the inner mitochondrial membrane. The human genome encodes multiple ANT isoforms that are expressed in a tissue-specific manner. Recently a novel germ cell-specific member of the ANT family, ANT4 (SLC25A31) was identified. Although it is known that targeted depletion of ANT4 in mice resulted in male infertility, the functional biochemical differences between ANT4 and other somatic ANT isoforms remain undetermined. To gain insight into ANT4, we expressed human ANT4 (hANT4) in yeast mitochondria. Unlike the somatic ANT proteins, expression of hANT4 failed to complement an AAC-deficient yeast strain for growth on media requiring mitochondrial respiration. Moreover, overexpression of hANT4 from a multi-copy plasmid interfered with optimal yeast growth. However, mutation of specific amino acids of hANT4 improved yeast mitochondrial expression and supported growth of the AAC-deficient yeast on non-fermentable carbon sources. The mutations affected amino acids predicted to interact with phospholipids, suggesting the importance of lipid interactions for function of this protein. Each mutant hANT4 and the somatic hANTs exhibited similar ADP/ATP exchange kinetics. These data define common and distinct biochemical characteristics of ANT4 in comparison to ANT1, 2 and 3 providing a basis for study of its unique adaptation to germ cells.

Figure 1. Strategy for introduction of hANTs into the AAC2 locus.

Step1: KAN-MX6 cassette at the AAC2 locus in AAC triple mutant yeast (TCY119) was replaced with URA3 to establish the parent stain (URA-AAC2). Step2: PCR-generated N-terminal AAC + hANTs ORF fragments (yNhANTs) were used for transformation of URA-AAC2, and transformants were selected on rich glucose media containing 5-FOA.

Figure 2. Characterization and growth of humanized-ANT yeast strains.

(A) Confirmation of hANT gene insertion by PCR-restriction fragment length analysis. AAC2 locus was PCR amplified by primers positioned 5′ and 3′ of ORF. The fragments were digested with BglII and EcoRI, and separated on a 2% agarose gel. (B) Growth of hANT yeast on complete glucose medium (YPD). (C) Growth of hANT yeast on complete ethanol-glycerol medium (YPEG). (D) Growth curves of hANT yeast in YPEG determined by turbidity (O.D.600).

Figure 3. Over-production of yNhANT4 by high-copy plasmid inhibits yeast cell growth.

Both yNhANT4 and yNhANT2 ORF were cloned into a high-copy plasmid and expressed under the control of the GAL1 promoter. Three independent isolates, each transformed with one of the indicated plasmids (Empty plasmid, yNhANT4, and yNhANT2) were streaked on medium as indicated. (A) glucose medium, (B) galactose medium, and (C) YPEG + 0.1% galactose.

Figure 4. Primary amino acid sequence alignment of various ANTs.

A30, P95 and S202 of hANT4 are indicated by arrow. Transmembrane domains are shown as shaded sequences.

Figure 5. Growth of various yNhANT4 mutant yeasts on nonfermentable carbon sources.

The hANT4 mutant alleles that complemented the aacΔ yeast were re-introduced into the parent yeast (URA-AAC2). The yeast strains were cultured in rich nonfermentable carbon source media (YPEG) at 30°C for the indicated time. Growth was monitored by turbidity (O.D.600).

Figure 6. Mutations increase mitochondrial ANT4 protein levels.

The V5-tag was introduced at the C-terminus of yNhANT4 in yeast strains as shown. All yeasts were cultured in rich glucose media (YPD). (A) hANT4 mRNA level determined by real time PCR. ALG9 expression level was used to normalize hANT4 expression. (B) hANT4 protein level in whole cells and isolated mitochondria was determined by western blotting using an anti-V5 epitope antibody. As a loading control, the membrane was immunostained with an anti-yeast porin antibody. WT: unmutated strains.

Figure 7. ADP/ATP exchange kinetics of yNhANT4 A30V in yeast mitochondria.

The exchange reaction was initiated by adding various concentrations of ADP to freshly isolated mitochondria. The initial linear part of the kinetic curve was used as the initial velocity for the ATP efflux rate. The initial velocity was plotted against the substrate concentration [free ADP] using a 4-parameter logistic fit to describe the data shown here.

Figure 8. Location of hANT4 mutation sites in the bovine ANT1 structure.

Based on primary amino acid sequence alignment (Fig. 4), A30, P95 and S202 of hANT4 correspond to A18, P83 and A190 of bovine ANT1, respectively. Those sites are highlighted in yellow on the bovine ANT1 structure (Protein Data Bank ID: 1OKC). The ANT1 crystal structure is oriented to show the inter membrane space at the top and the mitochondrial matrix at the bottom (A). Panel B is rotated 90 degrees about a horizontal axis in the plane of the page compared to panel A to show the ANT1 region facing the cytoplasm. Panel C shows how fatty acid detergent elements are in close proximity to a specific position in the ANT1 crystal structure (S202 in ANT4, A190 in ANT1).