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Further Characterization of the Human Peroxisomal C-Terminal Targeting

Signal Protein Import Receptor MARC FRANSEN," PETER MARWEN: EVELINE BAUMGART,"

JOHANNES C. T. VANHOOREN," GUY P. MANNAERTS," AND PAUL P. VAN VELDHOVEN"

"Katholieke Universiteit Leuven, Campus Gasthuisberg A fdeling Farmaco logie

bCentrum voor Menselijke Efelijkheid B-3000 Leuven, Belgium

The Zellweger syndrome, a generalized peroxisomal disorder, is believed to result from a defect in peroxisomal protein import. The import of various peroxisomal ma- trix proteins relies on the presence of a C-terminal tripeptide (serine-lysine-leucine, SKL) that acts as a peroxisomal targeting signal (PTSl). Using the yeast two-hybrid system, we identified and cloned a human cDNA encoding a protein that specifically bound PTSl I (PTS1-BP). The PTS1-BP is a 70-kDa polypeptide possessing tetratri- copeptide repeats and showing homology to the yeast PAS8 and PAS10 gene prod- ucts. By means of subcellular fractionation of human liver and immunofluorescence studies on HepG2 cells, the PTS 1 -BP was located to the cytosolic side of the peroxi- soma1 membrane.' Independently, Dodt et aL2 reported on a similar but slightly smaller gene product (PXRlp), probably the result of alternative splicing, that com- plemented group 2 of the generalized peroxisomal disorders. Overall, the studies by Dodt et a1.* and our studies' indicate that PXRlp and PTS1-BP are the PTSl protein import receptor.

Genomic Southern blotting indicated that the human PTSl receptor is probably encoded by a single copy gene. Fluorescence in situ hybridization on metaphase chromosome spreads from normal human white blood cells located the PTS 1 -recep- tor gene on chromosome 12~13. Refining the position of the PTSl receptor on the integrated chromosome 12 map by analysis of a subset of hybrids derived from a ra- diation hybrid panel for chromosome 12 indicated that the PTSl-receptor gene is flanked by TPIl and D12S1089. This localization will facilitate the detection and di- agnosis of peroxisome biogenesis disorder complementation in group 2 patients.

Biochemical characterization of the binding of the PTSl proteins to the receptor demonstrated that binding is optimal at pH 6.5 and is N-ethylmaleimide (NEM) sen- sitive. Wendland and Subramani3 reported the involvement of an NEM-sensitive pro- tein(s) in the peroxisomal protein import machinery. They demonstrated that this component(s) is a membrane-bound (or -associated) protein(s), and not a cytosolic protein(s). This result is in good agreement with our observation that the PTS 1 recep- tor is NEM sensitive and located to the cytosolic side of the peroxisomal membrane. It remains to be determined whether the sulfhydryl groups involved are part of the

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PTS1-binding site or are playing an indirect role in forming or stabilizing the tertiary structure of the PTSl receptor.

In addition, the C-terminal tripeptide SQL of the recently cloned pristanoyl-CoA oxidase4 was specifically recognized by the PTSl receptor. This indicates that SQL can also function as a peroxisomal targeting signal in mammalian cells and implies that, as in trypanosomes, lysine can be substituted not only by another basic amino acid such as histidine and arginine, but also by an amino acid capable of hydrogen bonding.s

REFERENCES

1. FRANSEN, M., C. BREES, E. BAUMGART, J. C. T. VANHOOREN, M. BAES, G. P. MANNAERTS & P.

2. DODT, G., N. BRAVERMAN, C . WONG, A. MOSER, H. W. MOSER, P. WATKINS, D. VALLE & S. J.

3. WENDLAND, M. & S. SUBRAMANI. 1993. J. Cell Biol. 120: 675-685. 4. VANHOOREN, J. C. T., M. FRANSEN, B. DE BETHUNE, E. BAUMCART, S. TORREKENS, M. BAES,

F. VAN LEUVEN, G. P. MANNAERTS & P. P. VAN VELDHOVEN. 1996. Eur. J. Biochem. 239:

5 . SOMMER, J. M., Q.-L. CHENG, G. A. KELLER & C. C. WANG. 1992. Mol. Biol. Cell 3:

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