The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with
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O R I GI N A L P A P E R
A. Gurvitz á L. Wabnegger á S. Langer B. Hamilton á H. Ruis á A. Hartig
The tetratricopeptide repeat domains of human, tobacco, and nematode PEX5 proteins are functionally interchangeable with the analogous native domain for peroxisomal import of PTS1-terminated proteins in yeast Received: 29 September 2000 / Accepted: 4 November 2000 / Published online: 19 January 2001 Ó Springer-Verlag 2001
Abstract In the yeast Saccharomyces cerevisiae, b-oxidation of fatty acids is compartmentalised in peroxisomes. Most yeast peroxisomal matrix proteins contain a type 1 C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL or a conservative variant thereof. PTS1-terminated proteins are imported by Pex5p, which interacts with the targeting signal via a tetratricopeptide repeat (TPR) domain. Yeast cells devoid of Pex5p are unable to import PTS1-containing proteins and cannot degrade fatty acids. Here, the PEX5TPR domains from human, tobacco, and nematode were inserted into a TPR-less yeast Pex5p construct to generate Pex5p chimaeras. These hybrid proteins were examined for functional complementation of the pex5D mutant phenotype. Expression of the Pex5p chimaeras in pex5D mutant cells restored peroxisomal import of PTS1terminated proteins. Chimaera expression also re-established degradation of oleic acid, allowing growth on this fatty acid as a sole carbon source. We conclude that, in the context of Pex5p chimaeras, the human, tobacco, and nematode Pex5p-TPR domains are functionally interchangeable with the native domain for the peroxisomal import of yeast proteins terminating with canonical PTS1s. Non-conserved yeast PTS1s, such as HRL and HKL, did not interact with the tobacco PEX5TPR domain in the two-hybrid system. HRL occurs at the C-terminus of the peroxisomal protein Eci1p, which is required for growth on unsaturated fatty acids. Although mutant pex5D cells expressing a yeast/tobacco Pex5p chimaera failed to import a GFP-Eci1p reporter protein, Communicated by C. P. Hollenberg A. Gurvitz á L. Wabnegger á S. Langer á B. Hamilton H. Ruis á A. Hartig (&) Vienna Biocenter, Institut fuÈr Biochemie und Molekulare Zellbiologie der UniversitaÈt Wien and Ludwig Boltzmann-Forschungsstelle fuÈr Biochemie, Dr Bohrgasse 9, 1030 Vienna, Austria E-mail: [email protected] Fax: 43-1-4277-9528
they were able to grow on oleic acid. We reason that this is due to a cryptic PTS in native Eci1p that can function in a redundant system with the C-terminal HRL. Key words Saccharomyces cerevisiae pex5D mutant á Peroxisome biogenesis á b-Oxidation á Unsaturated fatty acids á Eci1p
Introduction Peroxisomes are organelles that have a single limiting membrane and occur in all eukaryotic cells. The peroxisomal matrix characteristically contains enzymes for reactions involving molecular oxygen and for forming hydrogen peroxide (de Duve and Baudhuin 1966). In the yeast Saccharomyces cerevisiae, peroxisomes also represent the sole site for fatty acid b-oxidation (Kunau et al. 1995). Peroxisomal matrix
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