Mislocalization of melanosomal proteins in melanocytes from mice with oculocutaneous albinism type 2.

More than 10% of admissions worldwide to institutions for the visually impaired are due to some form of albinism. The most common form, oculocutaneous albinism type 2, results from mutations at the p locus. The function of the p gene is yet to be determined. It has been shown that melanocytes from p -null mice exhibit an abnormal melanosomal ultrastructure in addition to alterations in activity and localization of tyrosinase, a critical melanogenic enzyme. In light of these observations, we examined tyrosinase trafficking in p -null vs wildtype mouse melanocytes in order to explore p function. Electron microscopy of wildtype melan-a and p -null melan-p1 cells demonstrated accumulation of tyrosinase in 50 nm vesicles throughout the cell in the absence of p, an observation corroborated by an increase in tyrosinase activity in vesicle-enriched fractions from melan-p1 compared to melan-a cells. Misrouting in the absence of p was not limited to tyrosinase; a second melanosomal protein, tyrosinase-related protein 1, also trafficked incorrectly. In melan-p1, mislocalization led to secretion of tyrosinase into the medium. Adding tyrosine to the medium was found to partially correct tyrosinase trafficking and to reduce secretion; the cysteine protease inhibitor E64 also reduced secretion. We propose that p is required by melanocytes for transport of melanosomal proteins. In its absence, tyrosinase accumulates in vesicles and, in cultured melanocytes, is proteolysed and secreted.

High-molecular-weight forms of tyrosinase and the tyrosinase-related proteins: evidence for a melanogenic complex.

Tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2, (TRP-2, dopachrome tautomerase) were shown by immunoblotting and enzyme assays to copurify from extracts of Cloudman S91 melanoma cells. Antibodies to TRP-1 and TRP-2 immunoprecipitated tyrosinase activity, suggesting a stable interaction (complex) among these proteins. The tyrosine hydroxylase activity of tyrosinase was reduced in the complexed form; treatment with Triton X-100 dissociated the complex and activated the tyrosinase present within it. To further study this complex, we employed sucrose gradient density centrifugation of extracts from cultured murine melanocytes. Tyrosinase, TRP-1, and TRP-2 all existed in high molecular weight "multimers" of approximately 200 to > 700 kilodaltons. Extraction of cells with buffers containing the detergent CHAPS preserved the high molecular weight multimers; Triton X-100 caused their dissociation into monomers. Low pH, low ionic strength, and millimolar concentrations of calcium ions favored the maintenance of multimers. The results of this study demonstrate that the participation of tyrosinase, TRP-1, and TRP-2 in a multimeric complex could have important physiologic consequences, and raise the possibility that some of the well-known interactions between coat color genes may be explained by intermolecular interactions between the gene products.

Pathogenesis of the platinum (cp) mutation, a model for oculocutaneous albinism.

The platinum mutation at the C (albino) locus in the mouse is a potential model for oculocutaneous albinism in humans other than type IA (tyrosinase-negative) albinism. Although tissues from mice homozygous for the mutation display substantial tyrosinase activity, cutaneous and ocular pigmentation is severely restricted in affected animals. Using specific antipeptide antisera, we demonstrate that ocular extracts from wild-type mice contain two isoforms of tyrosinase bearing either the amino-terminal PEP5 epitope or the carboxy-terminal PEP7 epitope. The latter isoform participates in a high-molecular-weight complex detectable on sucrose density gradients. In platinum mice, antiserum to the PEP7 epitope fails to recognize any protein species, and the high-molecular-weight form of tyrosinase is not detectable. Our results support a key role for this high-molecular-weight complex in melanogenesis, and suggest that mutations that interfere with the ability of tyrosinase to participate in a multimeric complex may be a cause of oculocutaneous albinism in people.

Identification of a mammalian melanosomal matrix glycoprotein.

Antiserum raised in rabbits against the Triton X-100 insoluble fraction of melanosomes from mouse melanoma cells specifically decorates the internal matrix of melanosomes in immunoelectron microscopy. In metabolic labeling studies, the antiserum recognizes a protein of 94 kDa, which is processed to a band of 53 kDa. Whereas the precursor is relatively soluble in buffers containing Triton X-100, the processed protein requires the addition of sodium dodecyl sulfate for effective solubilization, as would be expected for a melanosomal matrix constituent. Tunicamycin reduces the Mr of the nascent protein to 75 kDa, but deoxymannojirimycin and swainsonine have no effect, suggesting that following initial glycosylation in the endoplasmic reticulum, the protein is not subject to processing by glycosidases in the Golgi apparatus or may bypass it entirely. Subcellular fractionation followed by immunoblotting confirms that the protein is present in the melanosome-rich, large granule fraction. Expression of the protein is regulated differently from that of the tyrosinase-related protein family. Conditions that greatly stimulate expression of tyrosinase-related proteins do not affect matrix protein expression, nor is the protein immunologically related to the tyrosinase-related protein family. Our results suggest that we have identified an authentic component of the mammalian melanosomal matrix, and that its characteristics lend support to a bipartite pathway for melanosomal biogenesis.

Lysosome-associated membrane protein-1 (LAMP-1) is the melanocyte vesicular membrane glycoprotein band II.

Coated vesicles play a critical role in the process of melanogenesis. Antisera raised against a coated vesicle fraction from mouse melanoma cells recognize two major glycoprotein antigens, band I (47-55 kd) and band II (90-120 kd). We demonstrate that band II is lysosome-associated membrane protein 1 (LAMP-1) by the following criteria: 1) the molecular weight and abundance of LAMP-1 varies among tissues but is always identical to that of band II; 2) band II and LAMP-1 co-migrate in sucrose gradient sedimentation studies; 3) immunodepletion of cell extracts with antivesicle serum removes all LAMP-1; and 4) intact organelles immunoisolated with antivesicle serum contain band II and LAMP-1. Our results further confirm the long-suspected relationship between melanosomes and the lysosomal lineage of organelles.

Subcellular distribution of tyrosinase and tyrosinase-related protein-1: implications for melanosomal biogenesis.

Are tyrosinase, encoded at the albino locus, and tyrosinase-related protein-1 (TRP-1), encoded at the brown locus, similarly distributed in melanocytes? We determined the subcellular distribution of tyrosinase and TRP-1 using density fractionation of postnuclear supernatants from mouse melanoma cells of defined genotype followed by immunoblotting with specific antipeptide sera. In highly melanized cells, the majority of tyrosinase cosedimented on Percoll density gradients with visible melanin and with the peak of DOPA incorporation, confirming its presence predominantly in stage III-IV melanosomes. In contrast, the distribution of TRP-1 was limited to a less-dense melanosomal compartment, devoid of melanin. In amelanotic or minimally melanized cells, the majority of tyrosinase shifted into these lighter peaks. To explore a suspected relationship between lysosomes and melanosomes, we analyzed the distribution of lysosome-associated membrane protein-1 (LAMP-1). An overlap in the distribution of LAMP-1 and TRP-1 was demonstrated by immunomicroscopy and confirmed by immunoisolation. LAMP-1 was not present in the dense, melanin-rich melanosomal peak on gradient analysis. TRP-1 from melanoma cells homozygous for the brown mutation is not fully glycosylated, is more rapidly degraded, and is restricted in its distribution compared to its wild-type counterpart. In these mutant cells, all melanosomal compartments contain LAMP-1. Our results demonstrate that in wild-type cells the majority of tyrosinase eventually localizes to stage III-IV melanosomes. TRP-1 is limited to a less dense melanosomal compartment that is also LAMP-1 positive. The existence of this compartment suggests that it may represent a common step in the biogenesis of melanosomes and lysosomes.

Infectious measles virus from cloned cDNA.

The study of measles virus (MV) and of negative strand RNA viruses in general has been hampered by the lack of an experimental system for genetic manipulation. Here we describe a procedure for generating infectious MV from cloned MV cDNA. First we assembled a genetically marked DNA copy of the MV genome in plasmids, under the control of phage T3 or T7 promoters, allowing production of transcripts almost identical to the MV genome or antigenome. Incubation of these linearized plasmid DNAs with the appropriate phage polymerase and only two ribonucleoside triphosphates yielded committed transcription complexes. Microinjection of these complexes into the cytoplasm of helper cells which provide the proteins necessary for MV genome encapsidation and transcription/replication, reproducibly give rise to lytic MVs. The transcripts of one of these viruses were analysed by sequencing after reverse transcription followed by DNA amplification, and found to contain the genetic tags. The described procedure permits the analysis of a negative strand RNA virus with the same genetic tools previously applicable only to positive strand RNA viruses and retroviruses.

Purification and distribution of a novel macrophage-specific calmodulin-binding glycoprotein.

The murine macrophage-like cell line J774.16 and peritoneal exudate cells elicited with thioglycollate or starch contain a major calmodulin (CaM)-binding protein (CaMBP) which is absent in trifluoperazine-resistant variants of J774, resident peritoneal macrophages and peritoneal macrophages elicited with concanavalin A, lipopolysaccharide, proteose peptone, or Bacillus Calmette-Guerin. Resident murine peritoneal cells maintained in tissue culture for 3 days begin to accumulate this protein, as do human peripheral blood monocytes after 7 days of culture. A specific competitive displacement radioimmunoassay was developed with the use of a rabbit antiserum raised to the partially purified CaM-binding protein and [125I]CaM covalently cross-linked to the principal CaM-binding protein in the preparation. The radioimmunoassay confirmed the unique cellular distribution of this protein, suggesting that it may be a marker for certain stages of macrophage differentiation. Monoclonal antibodies were prepared, and one of these was used to further purify the protein by immunoaffinity chromatography. A protein of Mr 50,000 to 60,000 was isolated. The protein could be selectively adsorbed to wheat germ agglutinin agarose and subsequently eluted with N-acetyl glucosamine. This property, plus the sensitivity of the protein to endoglycosidase F, led to the conclusion that it is a glycoprotein. The cellular distribution, subcellular localization, and evidence for glycosylation suggest that this protein may be a macrophage-specific receptor with a high affinity for Ca2+-CaM.

Trifluoperazine inhibits phagocytosis in a macrophagelike cultured cell line.

Trifluoperazine, a drug that binds to Ca2+-calmodulin and inhibits its interaction with other proteins, was found to inhibit growth and phagocytosis in a macrophagelike cell line, J774.16. Both effects were reversible and occurred at the same concentrations of drug (25--50 microM) that inhibited the activation of cyclic nucleotide phosphodiesterase by calmodulin in vitro. Fc-mediated phagocytosis was also depressed by W-7, a sulfonamide derivative that inhibits the activity of Ca2+-calmodulin. In contrast, taxol, a drug that stabilizes cellular microtubules, had no effect on Fc-mediated phagocytosis although it inhibited cell growth at nanomolar concentrations. The inhibitory effects of trifluoperazine and W-7 on phagocytosis suggest that calmodulin may be involved in this complex cellular function.