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. 1997 Aug 5;94(16):8509-14.
doi: 10.1073/pnas.94.16.8509.

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

F E Weber  1 , G Minestrini, J H Dyer, M Werder, D Boffelli, S Compassi, E Wehrli, R M Thomas, G Schulthess, H Hauser
Affiliations

Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily

F E Weber et al. Proc Natl Acad Sci U S A. .

Abstract

A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library. The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule. The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result. The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0. The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily. Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver. The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum. Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria. The Ca2+ binding N-terminal half of the transporter faces the cytosol.

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Figures

Figure 1
Figure 1
Schematic drawing of the protein and the comparison of the EF-hand motifs. (A) The location of the EF-hand motifs and the mitochondrial solute carrier structure are indicated. A scale for 100 amino acids is given. (B) The EF-hand sequences (EF) are shown in comparison to their consensus sequence drawn from ref. .
Figure 2
Figure 2
Expression of the Ca2+-dependent mitochondrial solute carrier in rabbit tissue. (A) Total RNA samples from nine segments from the small intestine; proximal (lane 1) through distal (lane 9), and (B) RNA samples from esophagus (lane 1), stomach (lane 2), liver (lane 3), spleen (lane 4), skeletal muscle (lane 5), heart (lane 6), lung (lane 7), testes (lane 8), brain (lane 9), kidney (lane 10), and colon (lane 11) were hybridized with the probe for the Ca2+-dependent mitochondrial solute carrier. The amounts of RNA were verified by visualizing the 28S and 18S rRNA bands in the agarose gels after staining with ethidium bromide (data not shown). The position of the 28S and 18S rRNA species are indicated.
Figure 3
Figure 3
Purity of the expressed N-terminal polypeptide and its ability to bind calcium. (A) Coomassie brilliant blue stained SDS/15% PAGE. Shown is E. coli lysate before (lane 1) and after induction (lane 2) with isopropyl β-d-thiogalactopyranoside. In lanes 3 and 4, 2 and 5 μg of purified fragments were loaded. (B) Autoradiograph of the proteins as shown in A transferred on a polyvinylidene difluoride membrane and labeled with 45Ca2+ as described.
Figure 4
Figure 4
Effect of Ca2+ on the near-UV (A) and far-UV (B) CD spectra of the Ca2+-dependent carrier. (A) The initial spectrum at zero free [Ca2+] was taken in 10 mM K2EGTA, 100 mM KCl, and 10 mM Mops (thick solid line). For the subsequent spectra, several mixtures containing different free calcium concentrations (0.15–7.34 μM) were used. These were prepared as described (20). (B) The same procedure was used to obtain the far-UV spectra at zero free [Ca2+] (thick solid line) and 40 μM free [Ca2+] (thin solid line).
Figure 5
Figure 5
Localization of the Ca2+- dependent solute carrier. Shown is the immunogold labeling of a section of an enterocyte of a villus from rabbit ileum (A) and a section through a sediment of a mitochondrial fraction (B). Immunogold label is found mainly in peroxisomes (per), but to some extent also in mitochondria (mi). Microsomal membranes (ms) carry no gold label (B). Freeze-substitution and LR-gold embedding have been described.
Figure 6
Figure 6
Topology of the Ca2+- dependent solute carrier. (A) Immunoblotting of 15 μg mitochondrial fraction protein before (lane 1) and after (lane 2) digestion with Arg-C protease is shown. The polyclonal antibodies were generated against a 25-kDa N-terminal polypeptide of the transporter. Note that with the disappearance of the 53-kDa transporter no proportional appearance of a fragment sized between 28 and 53 kDa occurs. Thus, externally administered proteases have access to the N terminus of the transporter. The apparent molecular masses of marker proteins are indicated; the procedure has been described in Materials and Methods. (B) Schematic drawing of a possible transmembrane arrangement of the Ca2+-dependent solute carrier based on our topology studies is shown. The EF-hand motifs of the Ca2+-binding N terminus are indicated. The model is based on the topology of the ADP/ATP transporter (34, 35).The N-terminal and C-terminal regions are exposed to the cytosol and the segment containing Arg-314 (R314) protrudes into the matrix. The size of the entire protein (53 kDa), the N terminus exposed to the cytosol (28 kDa), and of a fragment produced by cleavage at Arg-314 (38 kDa) are indicated.
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