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J. Biol. Chem., Vol. 280, Issue 6, 4817-4824, February 11, 2005

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Molecular Basis of the extreme dilution mottled Mouse Mutation

A COMBINATION OF CODING AND NONCODING GENOMIC ALTERATIONS^*

Alfonso Lavado, Concepción Olivares¶, José Carlos García-Borrón, and Lluís Montoliu||

From the Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología, Campus de Cantoblanco, C/Darwin 3, Madrid 28049 and the Department of Biochemistry and Molecular Biology and Immunology, School of Medicine, University of Murcia, Apto 4021, Campus Espinardo, Murcia 30100, Spain

Tyrosinase is the rate-limiting enzyme in melanin biosynthesis. It is an N-glycosylated, copper-containing transmembrane protein, whose post-translational processing involves intracytoplasmic movement from the endoplasmic reticulum to the Golgi and, eventually, to the melanosome. The expression of the tyrosinase (Tyr) gene is controlled by several regulatory regions including a locus control region (LCR) located 15 kb upstream from the promoter region. The extreme dilution mottled mutant mice (Tyr^c-em) arose spontaneously at the MRC Institute in Harwell (United Kingdom) from a chinchilla-mottled mutant (Tyr^c-m) stock, whose molecular basis corresponds to a rearrangement of 5'-upstream regulatory sequences including the LCR of the Tyr gene. Tyr^c-em mice display a variegated pigmentation pattern in coat and eyes, in agreement with the LCR translocation, but also show a generalized hypopigmented phenotype, not seen in Tyr^c-m mice. Genomic analyses of Tyr^c-em mice showed a C1220T nucleotide substitution within the Tyr encoding region, resulting in a T373I amino acid change, which abolishes an N-glycosylation sequon located in the second metal ion binding site of the enzyme. Tyrosinase from Tyr^c-em displayed a reduced enzymatic activity in vivo and in vitro, compared with wild-type enzyme. Deglycosylation studies showed that the mutant protein has an abnormal glycosylation pattern and is partially retained in the endoplasmic reticulum. We conclude that the phenotype of the extreme dilution mottled mouse mutant is caused by a combination of coding and noncoding genomic alterations resulting in several abnormalities that include suboptimal gene expression, abnormal protein processing, and reduced enzymatic activity.

Received for publication, September 10, 2004 , and in revised form, November 30, 2004.

This work was supported by Spanish Ministry of Science and Technology Grants Bio2000-1653 and Bio2003-08196, Comunidad Autónoma de Madrid 08.5/0046/2003 (to L. M.), and SAF2003-03411 from the Comisión Interministerial de Ciencia y Tecnologia and Fondo Europeo de Desarrollo Regional (to J. C. G.-B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY526904.

^¶ Present address: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 1 Avenue de la Terrasse, Gif-sur-Yvette 91198, France.

^|| To whom correspondence should be addressed. Tel.: 34-915-854-844; Fax: 34-915-854-506; E-mail: montoliu{at}cnb.uam.es.

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