The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (⌬5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and ⌬5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the ⌬5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin.Alternative mRNA splicing can potentially increase the number of proteins expressed by a single gene and may thus raise the complexity of the translated information. Although alternative splicing is common among human genes, evidence for the presence of multiple functionally active splice products has rarely been presented (1). Tafazzin is one example in which alternative splicing could, in theory, lead to a number of related proteins and where reasons for such diversification may exist.Tafazzin is a phospholipid-lysophospholipid transacylase (2) that is involved in the generation of the characteristic fatty acid profile 3 of mitochondrial cardiolipin (CL) 4 (3, 4) and that is mutated in patients with Barth syndrome (5), a human disease presenting with cardiomyopathy, skeletal muscle weakness, episodic neutropenia, growth disturbances, and abnormal mitochondria (6 -8). The initial study of the human tafazzin gene suggested that it produces 10 different mRNAs as a result of alternative initiation and splicing (5), but only four were confirmed by later studies (9, 10). Sequence alignment analysis demonstrated that hTAZ-⌬5, the splice variant that skips the fifth exon, shows the highest degree of homology to tafazzins from other organisms and thus likely represents the principal isoform (11). This idea was supported by the fact that hTAZ-⌬5 forms the dominant band in reverse ...

Proteomic analyses of the nucleolus have revealed almost 700 functionally diverse proteins implicated in ribosome biogenesis, nucleolar assembly, and regulation of vital cellular processes. However, this nucleolar inventory has not unveiled a specific consensus motif necessary for nucleolar binding. The ribosomal protein family characterized by their basic nature should exhibit distinct binding sequences that enable interactions with the rRNA precursor molecules facilitating subunit assembly. We succeeded in delineating 2 minimal nucleolar binding sequences of human ribosomal protein S6 by fusing S6 cDNA fragments to the 5' end of the LacZ gene and subsequently detecting the intracellular localization of the beta-galactosidase fusion proteins. Nobis1 (nucleolar binding sequence 1), comprising of 4 highly conserved amino acid clusters separated by glycine or proline, functions independently of the 3 authentic nuclear localization signals (NLSs). Nobis2 consists of 2 conserved peptide clusters and requires the authentic NLS2 in its native context. Similarly, we deduced from previous publications that the single Nobis of ribosomal protein S25 is also highly conserved. The functional protein domain organization of the ribosomal protein S6e family consists of 3 modules: NLS, Nobis, and the C-terminal serine cluster of the phosphorylation sites. This modular structure is evolutionary conserved in vertebrates, invertebrates, and fungi. Remarkably, nucleolar binding sequences of small and large ribosomal proteins reside in peptide clusters conserved over millions of years.

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