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. 2005 Aug 1;389(Pt 3):647-55.
doi: 10.1042/BJ20050283.

Novel variants of human SCaMC-3, an isoform of the ATP-Mg/P(i) mitochondrial carrier, generated by alternative splicing from 3'-flanking transposable elements

Araceli Del Arco  1
Affiliations

Novel variants of human SCaMC-3, an isoform of the ATP-Mg/P(i) mitochondrial carrier, generated by alternative splicing from 3'-flanking transposable elements

Araceli Del Arco. Biochem J. .

Abstract

CaMCs (calcium-dependent mitochondrial carriers) represent a novel subfamily of metabolite carriers of mitochondria. The ATP-Mg/P(i) co-transporter, functionally characterized more than 20 years ago, has been identified to be a CaMC member. There are three isoforms of the ATP-Mg/P(i) carrier in mammals, SCaMC-1 (short CaMC-1), -2 and -3 (or APC-1, -3 and -2 respectively), corresponding to the genes SLC25A24, SLC25A25 and SLC25A23 respectively, as well as six N-terminal variants generated by alternative splicing for SCaMC-1 and -2 isoforms. In the present study, we describe four new variants of human SCaMC-3 generated by alternative splicing. The new mRNAs use the exon 9 3'-donor site and distinct 5'-acceptor sites from repetitive elements, in regions downstream of exon 10, the last exon in all SCaMCs. Transcripts lacking exon 10 (SCaMC-3b, -3b', -3c and -3d) code for shortened proteins lacking the last transmembrane domain of 422, 456 and 435 amino acids, and were found in human tissues and HEK-293T cells. Mitochondrial targeting of overexpressed SCaMC-3 variants is incomplete. Surprisingly, the import impairment is overcome by removing the N-terminal extension of these proteins, suggesting that the hydrophilic N-terminal domain also participates in the mitochondrial import process, as shown for the CaMC members aralar and citrin [Roesch, Hynds, Varga, Tranebjaerg and Koehler (2004) Hum. Mol. Genet. 13, 2101-2111].

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Figures

Figure 1
Figure 1. Human SCaMC-3 gene exhibits alternative spliced variants derived from 3′-flanking repetitive elements
(A) Schematic representation of the different 3′ SCaMC-3 sequences found in databases. Black boxes indicate the common 5′-region, the novel 3′-end common to all SCaMC-3 variant analyses is indicated by an open box, and inserted sequences are indicated by stripes (accession numbers are shown). Numbered arrows indicate the relative position of selected specific primers used to amplify the different 3′-ends (described in the Materials and methods section). (B) RT–PCR analysis of SCaMC-3 3′-end variants. Reverse transcription was performed from total RNA of HEK-293T cells. The primer pairs used for each amplification are indicated at the top of each lane. The amplified products were verified by cloning and sequencing. The sizes of molecular-mass standards, HindIII-digested phage Φ29, are indicated. (C) Genomic organization of the 3′-region of the human SCaMC-3 gene. The genomic map was constructed from overlapping human BAC clones AC011539 and AC010503. The common exon 9 is shown as a black box. The grey box indicates the previously described SCaMC-3 last exon, exon 10, and the MaLR element is shown as an open box. Introns are shown as thick lines connecting these elements. Dark arrows represent intronic Alu elements (280 bp) and short arrows indicate truncated Alu sequences (120–150 bp), at their relative positions. The direction of the arrow shows the orientation of the inserted Alu element. Splicing events between upstream exon 9 and alternative 3′-exonized sequences are indicated by solid lines. (D) Alignment of the MLT1f consensus and that of novel SCaMC-3 3′-end nucleotide sequences. Only aligned regions with relevant homology are shown. Identical bases are indicated by asterisks. The MLT1f sequence is a fragment of a 944 bp-long consensus sequence deposited in RepBase Uptake [48]; nucleotide positions in the aligned MLT1f fragment are shown. The 2089 bp BC001656 nucleotide sequence comes from GenBank®. The polyadenylation signals are boxed.
Figure 2
Figure 2. Expression analysis of SCaMC-3b 3′-end variants in human tissues
Equivalent aliquots of a multiple human cDNA panel; pancreas, liver, kidney, heart, placenta, lung, skeletal (Sh.) muscle and brain (ClonTech) were used as templates. The upper panel shows the results obtained with primers specific for SCaMC-3a transcript, and the bottom one the PCR products co-amplified with primers 9 and 10b, corresponding to SCaMC-3b, -3b′, -3c and -3d transcripts. The sizes of amplified fragments are given in terms of bp. The products obtained were verified by sequencing. The results indicate a similar distribution pattern of SCaMC-3a and transcripts containing repetitive elements.
Figure 3
Figure 3. Alternative 3′-SCaMC-3 transcripts encoding for C-terminal truncated variants
(A) Upper panel: splicing acceptor sites involved in the generation of alternative SCaMC-3b and -3b′ transcripts. Genomic sequences derive from BAC clones mentioned in Figure 1. Alternative 3′-splice sites, site 1 and site 2, are shown. Lower panels: predicted amino acid sequences for the C-termini of SCaMC-3b and SCaMC-3b′ are shown. Exonic sequences from exon 9 are indicated in italic bold face and the sequence derived from MLT1f element by normal letters. Additional nucleotides in SCaMC-3b′ transcript are boxed. Predicted position of the TM-5 domain and equivalence with MC signature are shown. (B) The 3′-end sequence of SCaMC-3d transcript and its putative ORF, identical with SCaMC-3c, are shown at the top. Sequences from exon 9 and MLT1f element are represented as in (A). Alu-derived nucleotides are double-underlined and additional SCaMC-3d-specific sequences are indicated by the broken line.
Figure 4
Figure 4. Subcellular distribution of overexpressed SCaMC-3a, SCaMC-3b and SCaMC-3c/-3d variants
(A) Immunofluorescence assays of COS-7 cells transiently transfected with FLAG-tagged SCaMC-3 variants. Cells were transfected with the indicated constructs, labelled with MitoTracker, processed using an anti-FLAG monoclonal antibody and visualized with FITC secondary antibody. Two different patterns (I and II) were detected. Identical fields labelled for MitoTracker and anti-FLAG for representative cells of each pattern are presented. All images are processed in parallel. Magnification, ×100. (B) Mitochondrial localization of SCaMC proteins with N-terminal deletions (ΔNT-SCaMCs). COS-7 cells were transfected with ΔNT-SCaMC-3–FLAG constructs (ΔNT-SCaMC-3a, ΔNT-SCaMC-3b and ΔNT-SCaMC-3c/-3d) coding for the MC homology domain tagged at its C-terminus with FLAG. The cells were labelled with MitoTracker and with anti-FLAG antibody as in (A). (C) Western-blot analysis of overexpressed SCaMC isoforms in mitochondrial-enriched extracts (MIT) and post-mitochondrial (Post-MIT) supernatants from HEK-293 cells. Protein (10 μg) of extracts from cells transiently transfected with the indicated constructs were analysed. Membranes were immunoblotted with anti-FLAG antibody and reblotted with polyclonal anti-SCaMC-1 to test for mitochondrial fractionation. Sizes of immunoreactive anti-FLAG bands are coincident with the expected ones. Positions of molecular-mass standards are indicated.
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