Hermansky-Pudlak syndrome (HPS) is an often-fatal autosomal recessive disease in which albinism, bleeding, and lysosomal storage result from defects of diverse cytoplasmic organelles: melanosomes, platelet dense bodies, and lysosomes. HPS is the most common single-gene disorder in Puerto Rico, with an incidence of 1 in 1,800. We have identified the HPS gene by positional cloning, and found homozygous frameshifts in this gene in Puerto Rican, Swiss, Irish and Japanese HPS patients. The HPS polypeptide is a novel transmembrane protein that is likely to be a component of multiple cytoplasmic organelles and that is apparently crucial for their normal development and function. The different clinical phenotypes associated with the different HPS frameshifts we observed suggests that differentially truncated HPS polypeptides may have somewhat different consequences for subcellular function.

Although deficiencies in the retromer sorting pathway have been linked to late-onset Alzheimer's disease, whether these deficiencies underlie the disease remains unknown. Here we characterized two genetically modified animal models to test separate but related questions about the effects that retromer deficiency has on the brain. First, testing for cognitive defects, we investigated retromer-deficient mice and found that they develop hippocampaldependent memory and synaptic dysfunction, which was associated with elevations in endogenous A␤ peptide. Second, testing for neurodegeneration and amyloid deposits, we investigated retromer-deficient flies expressing human wild-type amyloid precursor protein (APP) and human ␤-site APP-cleaving enzyme (BACE) and found that they develop neuronal loss and human A␤ aggregates. By recapitulating features of the disease, these animal models suggest that retromer deficiency observed in late-onset Alzheimer's disease can contribute to disease pathogenesis.flies ͉ mice ͉ pathophysiology

Hermansky-Pudlak syndrome (HPS) is a rare, autosomal recessive disorder in which oculocutaneous albinism, bleeding, and lysosomal ceroid storage result from defects of multiple cytoplasmic organelles-melanosomes, platelet-dense granules, and lysosomes. As reported elsewhere, we mapped the human HPS gene to chromosome segment 10q23, positionally cloned the gene, and identified three pathologic mutations of the gene, in patients from Puerto Rico, Japan, and Europe. Here, we describe mutation analysis of 44 unrelated Puerto Rican and 24 unrelated non-Puerto Rican HPS patients. A 16-bp frameshift duplication, the result of an apparent founder effect, is nearly ubiquitous among Puerto Rican patients. A frameshift at codon 322 may be the most frequent HPS mutation in Europeans. We also describe six novel HPS mutations: a 5' splice-junction mutation of IVS5, three frameshifts, a nonsense mutation, and a one-codon in-frame deletion. These mutations define an apparent frameshift hot spot at codons 321-322. Overall, however, we detected mutations in the HPS gene in only about half of non-Puerto Rican patients, and we present evidence that suggests locus heterogeneity for HPS.

Mutations in the P gene of humans and the homologous p-locus of mice, respectively, result in the homologous disorders oculocutaneous albinism type 2 (OCA2) and pink-eyed dilution. Although clearly required for melanin biosynthesis, the specific function of the P gene product, a melanosomal transmembrane protein expressed in melanocytes of the skin, hair, and eyes, is not yet known. Here we describe lines of immortal melanocytes and melanoblasts from mice of the null genotype p(cp)/p(25H). These p-null melanocytes were severely hypopigmented, although they and the melanoblasts expressed mRNAs for a number of melanosomal proteins. Proliferation of the p-null melanoblasts was normal. Both diploid and immortal p-null melanocytes grew more slowly than wild-type melanocytes, however, and were unusually susceptible to the antibiotic G418; these abnormalities were corrected by culture in high concentrations of L-tyrosine. Transfection of the p-null melanocytes with full-length normal human P cDNA resulted in complementation of deficient melanin biosynthesis and hypopigmentation. In contrast, transfection with mutant human P cDNAs containing amino acid substitutions (A481T, V443I) found in patients with OCA2 resulted in minimal or partial correction, consistent with the corresponding pigmentation phenotypes in patients with these mutations. These results demonstrate the utility of this model system for distinguishing true OCA2 mutations from nonpathologic polymorphisms and for quantitating the effect of these mutations on P function.

Tyrosinase (EC 1.14.18.1) is a copper-containing enzyme that catalyzes several reactions in the biosynthesis of melanin pigments and is deficient in patients with type I oculocutaneous albinism (OCA1). Tyrosinase is thought to bind two copper ions, one at each of two conserved sequence motifs, termed CuA and CuB, but to date this has been directly proved only for the Neurospora and mushroom enzyme. Here, we demonstrate that mammalian tyrosinase directly binds copper, and that the CuA and CuB sites are both required for copper binding and for catalytic activity. We show that in human tyrosinase, copper binding by the CuB site is most likely coordinated by residues His363, His367, and His389, and that copper binding may be cooperative, with copper binding at one site facilitating copper binding by the other site. Furthermore, correct folding of the tyrosinase polypeptide appears to be necessary for copper binding, and a number of human OCA1 mutations disrupt copper binding and thus catalytic function of tyrosinase.