split-doa10 a naturally split polytopic eukaryotic membrane protein generated by fission of a nuclear genesplit-doa10自然分割多面体真核膜蛋白所产生的裂变核基因.pdfVIP

Split-Doa10: A Naturally Split Polytopic Eukaryotic Membrane Protein Generated by Fission of a Nuclear Gene 1 1¤ 2 1 Elisabeth Stuerner , Shigehiro Kuraku , Mark Hochstrasser , Stefan G. Kreft * 1 Department of Biology, University of Konstanz, Konstanz, Germany, 2 Department of Molecular Biophysics Biochemistry, Yale University, New Haven, Connecticut, United States of America Abstract Large polytopic membrane proteins often derive from duplication and fusion of genes for smaller proteins. The reverse process, splitting of a membrane protein by gene fission, is rare and has been studied mainly with artificially split proteins. Fragments of a split membrane protein may associate and reconstitute the function of the larger protein. Most examples of naturally split membrane proteins are from bacteria or eukaryotic organelles, and their exact history is usually poorly understood. Here, we describe a nuclear-encoded split membrane protein, split-Doa10, in the yeast Kluyveromyces lactis. In most species, Doa10 is encoded as a single polypeptide with 12–16 transmembrane helices (TMs), but split-KlDoa10 is encoded as two fragments, with the split occurring between TM2 and TM3. The two fragments assemble into an active ubiquitin-protein ligase. The K. lactis DOA10 locus has two ORFs separated by a 508-bp intervening sequence (IVS). A promoter within the IVS drives expression of the C-terminal KlDoa10 fragment. At least four additional Kluyveromyces species contain an IVS in the DOA10 locus, in contrast to even closely related genera, allowing dating of the fission event to the base of the genus. The upstream Kluyveromyces Doa10 fragment with its N-terminal RING-CH and two TMs resembl