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Ultraviolet (UV) light and incompletely understood genetic and epigenetic variations determine skin color. Here we describe an UV- and microphthalmia-associated transcription factor (MITF)-independent mechanism of skin pigmentation. Targeting the mitochondrial redox-regulating enzyme nicotinamide nucleotide transhydrogenase (NNT) resulted in cellular redox changes that affect tyrosinase degradation. These changes regulate melanosome maturation and, consequently, eumelanin levels and pigmentation. Topical application of small-molecule inhibitors yielded skin darkening in human skin, and mice with decreased NNT function displayed increased pigmentation. Additionally, genetic modification of NNT in zebrafish alters melanocytic pigmentation. Analysis of four diverse human cohorts revealed significant associations of skin color, tanning, and sun protection use with various single-nucleotide polymorphisms within NNT. NNT levels were independent of UVB irradiation and redox modulation. Individuals with postinflammatory hyperpigmentation or lentigines displayed decreased skin NNT levels, suggesting an NNT-driven, redox-dependent pigmentation mechanism that can be targeted with NNT-modifying topical drugs for medical and cosmetic purposes., Competing Interests: Declaration of interests D.E.F. and E.R. have a patent filed on “Methods and compositions for enhancing skin pigmentation” (publication number WO/2016/077817, May 19, 2016.). D.E.F. has a financial interest in Soltego, Inc., a company developing SIK inhibitors for topical skin darkening treatments that might be used for a broad set of human applications. D.E.F.’s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict-of-interest policies. B.P.K. is an inventor on patents and patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. consults for Avectas Inc., ElevateBio, and EcoR1 capital and is an advisor to Acrigen Biosciences. Q.Y.W. is a shareholder in Mymiel Skincare. L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, and Scholar Rock. He is a consultant for Celularity and Cellarity. H.W. is an employee and shareholder of Johnson and Johnson., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease., Competing Interests: This study was funded by Ajinomoto Co., Inc., Japan and one of the authors [M.B.] is an employee of this commercial funder. No other employment or consultancy relationships exist with the commercial funder, and no patents, products in development, or marketed products result from this study. The authors declare that no competing interests exist and that the commercial affiliation of one of the authors does not alter the adherence of authors to all PLOS ONE policies on sharing data and materials.

Lysosomes and mitochondria occupy a central stage in the maintenance of cellular homeostasis, by playing complementary roles in nutrient sensing and energy metabolism. Specifically, these organelles function as signaling hubs that integrate environmental and endogenous stimuli with specific metabolic responses. In particular, they control various lipid biosynthetic and degradative pipelines, either directly or indirectly, by regulating major cellular metabolic pathways, and by physical and functional connections established with each other and with other organelles. Membrane contact sites allow the exchange of ions and molecules between organelles, even without membrane fusion, and are privileged routes for lipid transfer among different membrane compartments. These inter-organellar connections typically involve the endoplasmic reticulum. Direct membrane contacts have now been described also between lysosomes, autophagosomes, lipid droplets, and mitochondria. This review focuses on these recently identified membrane contact sites, and on their role in lipid biosynthesis, exchange, turnover and catabolism. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Little is known as to how cells ensure that organelle size and number are coordinated to correctly couple organelle biogenesis to the demands of proliferation or differentiation. OA1 is a melanosome-associated G-protein-coupled receptor involved in melanosome biogenesis during melanocyte differentiation. Cells lacking OA1 contain fewer, but larger, mature melanosomes. Here, we show that OA1 loss of function reduces both the basal expression and the α-melanocyte-stimulating hormone/cAMP-dependent induction of the microphthalmia-associated transcription factor (MITF), the master regulator of melanocyte differentiation. In turn, this leads to a significant reduction in expression of PMEL, a major melanosomal structural protein, but does not affect tyrosinase and melanin levels. In line with its pivotal role in sensing melanosome maturation, OA1 expression rescues melanosome biogenesis, activates MITF expression and thereby coordinates melanosome size and number, providing a quality control mechanism for the organelle in which resides. Thus, resident sensor receptors can activate a transcriptional cascade to specifically promote organelle biogenesis., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Membrane contact sites (MCSs) allow the exchange of molecules and information between organelles, even when their membranes cannot fuse directly. In recent years, a number of functions have been attributed to these contacts, highlighting their critical role in cell homeostasis. Although inter-organellar connections typically involve the endoplasmic reticulum (ER), we recently reported the presence of a novel MCSs between melanosomes and mitochondria. Melanosome-mitochondrion contacts appear mediated by fibrillar bridges resembling the protein tethers linking mitochondria and the ER, both for their ultrastructural features and the involvement of Mitofusin 2. The frequency of these connections correlates spatially and timely with melanosome biogenesis, suggesting a functional link between the 2 processes and in general that organelle biogenesis in the secretory pathway requires interorganellar crosstalks at multiple steps. Here, we summarize the different functions attributed to MCSs, and discuss their possible relevance for the newly identified melanosome-mitochondrion liaison.

Background: To efficiently supply ATP to sites of high-energy demand and finely regulate calcium signaling, mitochondria adapt their metabolism, shape, and distribution within the cells, including relative positioning with respect to other organelles. However, physical contacts between mitochondria and the secretory/endocytic pathway have been demonstrated so far only with the ER, through structural and functional interorganellar connections., Results: Here we show by electron tomography that mitochondria physically contact melanosomes, specialized lysosome-related organelles of pigment cells, through fibrillar bridges resembling the protein tethers linking mitochondria and the ER. Mitofusin (Mfn) 2, which bridges ER to mitochondria, specifically localizes also to melanosome-mitochondrion contacts, and its knockdown significantly reduces the interorganellar connections. Contacts are associated to the melanogenesis process, as indicated by the fact that they are reduced in a model of aberrant melanogenesis whereas they are enhanced both where melanosome biogenesis takes place in the perinuclear area and when it is actively stimulated by OA1, a G protein-coupled receptor implicated in ocular albinism and organellogenesis. Consistently, Mfn2 knockdown prevents melanogenesis activation by OA1, and the pharmacological inhibition of mitochondrial ATP synthesis severely reduces contact formation and impairs melanosome biogenesis, by affecting in particular the developing organelles showing the highest frequency of contacts., Conclusions: Altogether, our findings reveal the presence of an unprecedented physical and functional connection between mitochondria and the secretory/endocytic pathway that goes beyond the ER-mitochondria linkage and is spatially and timely associated to secretory organelle biogenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Multivesicular endosomes/bodies (MVBs) deliver proteins, such as activated EGF receptor (EGFR), to the lysosome for degradation, and, in pigmented cells, MVBs containing PMEL are an initial stage in melanosome biogenesis. The mechanisms regulating numbers and fate of different populations of MVB are unclear. Here, we focus on the role of the G-protein-coupled receptor OA1 (also known as GPR143), which is expressed exclusively in pigmented cells and mutations in which cause the most common type of ocular albinism. When exogenously expressing PMEL, HeLa cells have been shown to form MVBs resembling early stage melanosomes. To focus on the role of OA1 in the initial stages of melanosome biogenesis we take advantage of the absence of the later stages of melanosome maturation in HeLa cells to determine whether OA1 activity can regulate MVB number and fate. Expression of wild-type but not OA1 mutants carrying inactivating mutations or deletions causes MVB numbers to increase. Whereas OA1 expression has no effect on delivery of EGFR-containing MVBs to the lysosome, it inhibits the lysosomal delivery of PMEL and PMEL-containing MVBs accumulate. We propose that OA1 activity delays delivery of PMEL-containing MVBs to the lysosome to allow time for melanin synthesis and commitment to melanosome biogenesis.

The epigenetic silencing of exogenous transcriptional units integrated into the genome represents a critical problem both for long-term gene therapy efficacy and for the eradication of latent viral infections. We report here that limitation of essential amino acids, such as methionine and cysteine, causes selective up-regulation of exogenous transgene expression in mammalian cells. Prolonged amino acid deprivation led to significant and reversible increase in the expression levels of stably integrated transgenes transcribed by means of viral or human promoters in HeLa cells. This phenomenon was mediated by epigenetic chromatin modifications, because histone deacetylase (HDAC) inhibitors reproduced starvation-induced transgene up-regulation, and transcriptome analysis, ChIP, and pharmacological and RNAi approaches revealed that a specific class II HDAC, namely HDAC4, plays a critical role in maintaining the silencing of exogenous transgenes. This mechanism was also operational in cells chronically infected with HIV-1, the etiological agent of AIDS, in a latency state. Indeed, both amino acid starvation and pharmacological inhibition of HDAC4 promoted reactivation of HIV-1 transcription and reverse transcriptase activity production in HDAC4(+) ACH-2 T-lymphocytic cells but not in HDAC4(-) U1 promonocytic cells. Thus, amino acid deprivation leads to transcriptional derepression of silenced transgenes, including integrated plasmids and retroviruses, by a process involving inactivation or down-regulation of HDAC4. These findings suggest that selective targeting of HDAC4 might represent a unique strategy for modulating the expression of therapeutic viral vectors, as well as that of integrated HIV-1 proviruses in latent reservoirs without significant cytotoxicity.

Melanosomes are the specialized intracellular organelles of pigment cells devoted to the synthesis, storage and transport of melanin pigments, which are responsible for most visible pigmentation in mammals and other vertebrates. As a direct consequence, any genetic mutation resulting in alteration of melanosomal function, either because affecting pigment cell survival, migration and differentiation, or because interfering with melanosome biogenesis, transport and transfer to keratinocytes, is immediately translated into color variations of skin, fur, hair or eyes. Thus, over 100 genes and proteins have been identified as pigmentary determinants in mammals, providing us with a deep understanding of this biological system, which functions by using mechanisms and processes that have parallels in other tissues and organs. In particular, many genes implicated in melanosome biogenesis have been characterized, so that melanosomes represent an incredible source of information and a model for organelles belonging to the secretory pathway. Furthermore, the function of melanosomes can be associated with common physiological phenotypes, such as variation of pigmentation among individuals, and with rare pathological conditions, such as albinism, characterized by severe visual defects. Among the most relevant mechanisms operating in melanosome biogenesis are the signal transduction pathways mediated by two peculiar G protein-coupled receptors: the melanocortin-1 receptor (MC1R), involved in the fair skin/red hair phenotype and skin cancer; and OA1 (GPR143), whose loss-of-function results in X-linked ocular albinism. This review will focus on the most recent novelties regarding the functioning of these two receptors, by highlighting emerging signaling mechanisms and general implications for cell biology and pathology., (Copyright 2010 Elsevier Ltd. All rights reserved.)

G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization. Enhancing beta2 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and delta opioid receptors. Furthermore, co-immunoprecipitation experiments showed that G15 alpha subunit (as opposed to Galphaq and Galphas) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by beta-arrestin. Interestingly, co-expression of Galpha15 partially rescued cell surface localization and signalling capabilities of the same mutant receptor and reduced beta2 adrenergic receptor internalization. Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters.

Oculocutaneous albinism type 2 is caused by defects in the gene OCA2, encoding a pigment cell-specific, 12-transmembrane domain protein with homology to ion permeases. The function of the OCA2 protein remains unknown, and its subcellular localization is under debate. Here, we show that endogenous OCA2 in melanocytic cells rapidly exits the endoplasmic reticulum (ER) and thus does not behave as a resident ER protein. Consistently, exogenously expressed OCA2 localizes within melanocytes to melanosomes, and, like other melanosomal proteins, localizes to lysosomes when expressed in nonpigment cells. Mutagenized OCA2 transgenes stimulate melanin synthesis in OCA2-deficient cells when localized to melanosomes but not when specifically retained in the ER, contradicting a proposed primary function for OCA2 in the ER. Steady-state melanosomal localization requires a conserved consensus acidic dileucine-based sorting motif within the cytoplasmic N-terminal region of OCA2. A second dileucine signal within this region confers steady-state lysosomal localization in melanocytes, suggesting that OCA2 might traverse multiple sequential or parallel trafficking routes. The two dileucine signals physically interact in a differential manner with cytoplasmic adaptors known to function in trafficking other proteins to melanosomes. We conclude that OCA2 is targeted to and functions within melanosomes but that residence within melanosomes may be regulated by secondary or alternative targeting to lysosomes.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport.

The protein product of the gene responsible for ocular albinism type 1, named OA1, is a pigment-cell-specific membrane glycoprotein, displaying features of G-protein-coupled receptors, yet exclusively localized to late endosomes, lysosomes and melanosomes. To dissect the signals responsible for the intracellular localization of OA1, we generated chimeric proteins consisting of the cytosolic domains of OA1 fused to the lumenal and transmembrane domains of LAMP1; in addition, we generated missense and deletion mutants of full-length OA1. Using this approach, we identified two separate sorting signals that are both necessary and sufficient for intracellular retention, as well as lysosomal and melanosomal localization, in melanocytic and non-melanocytic cells. These sorting signals are an unconventional dileucine motif within the third cytosolic loop and a novel motif, characterized by a tryptophan-glutamic acid doublet, within the C-terminal tail. Both motifs must be mutated to promote the plasma membrane localization of OA1, suggesting that they can independently drive its intracellular targeting. In addition, both motifs act similarly as lysosomal sorting signals in non-melanocytic cells, but appear to carry different specificities in melanocytic cells. Our findings indicate that OA1 contains multiple unconventional signals responsible for its lysosomal and melanosomal localization, and reveal a remarkable and unforeseen complexity in the regulation of polytopic protein sorting to specialized secretory organelles.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific integral membrane glycoprotein, localized to melanosomes and lysosomes and possibly implicated in melanosome biogenesis. Although its function remains unknown, we previously showed that OA1 shares structural similarities with G protein-coupled receptors (GPCRs). To ascertain the molecular function of OA1 and in particular its nature as a GPCR, we adopted a heterologous expression strategy commonly exploited to demonstrate GPCR-mediated signaling in mammalian cells. Here we show that when expressed in COS7 cells OA1 displays a considerable and spontaneous capacity to activate heterotrimeric G proteins and the associated signaling cascade. In contrast, OA1 mutants carrying either a missense mutation or a small deletion in the third cytosolic loop lack this ability. Furthermore, OA1 is phosphorylated and interacts with arrestins, well-established multifunctional adaptors of conformationally active GPCRs. In fact, OA1 colocalizes and coprecipitates with arrestins, which downregulate the signaling of OA1 by specifically reducing its expression levels. These findings indicate that heterologously expressed OA1 exhibits two fundamental properties of GPCRs, being capable to activate heterotrimeric G proteins and to functionally associate with arrestins, and provide proof of principle that OA1 can actually function as a canonical GPCR in mammalian cells.

Ocular albinism type 1 is an X-linked disorder characterized by severe reduction of visual acuity, retinal hypopigmentation, foveal hypoplasia, optic misrouting and the presence of giant melanosomes (macromelanosomes) in skin melanocytes and retinal pigment epithelium. The protein product of the OA1 gene is a pigment cell specific membrane glycoprotein, displaying structural and functional features of G protein-coupled receptors (GPCRs). However, in contrast to all other previously characterized GPCRs, OA1 is not localized to the plasma membrane, but is targeted to intracellular organelles, namely late endosomes/lysosomes and melanosomes. These unique characteristics suggest that OA1 represents the first example described so far of an exclusively intracellular GPCR and regulates melanosome biogenesis by transducing signals from the organelle lumen to the cytosol. These findings support previous hypotheses that GPCR-mediated signaling might also operate at the internal membranes in mammalian cells.

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by congenital nystagmus and photodysphoria, moderate to severe reduction of visual acuity, hypopigmentation of the retina, and the presence of macromelanosomes in the skin and eyes. We have previously isolated the gene for OA1 and characterized its protein product as melanosomal membrane glycoprotein displaying structural and functional features of G protein-coupled receptors. We and others have identified mutations of various types within the OA1 gene in patients with this disorder, including deletions and splice site, frameshift, nonsense, and missense mutations. However, different prevalences of large intragenic deletions have been reported, ranging from 10% to 50% in independent studies. To determine whether these differences might be related to the geographic origin of the OA1 families tested, we performed a further extensive mutation analysis study leading to the identification of pathogenic mutations in 30 unrelated OA1 patients mainly from Europe and North America. These results, together with our earlier mutation reports on OA1, allow us to resolve the apparent discrepancies between previous studies and point to a substantial difference in the frequency of large intragenic deletions in European (<10%) compared with North American (>50%) OA1 families. These observations and our overall refinement of point mutation distribution within the OA1 gene have important implications for the molecular diagnosis of OA1 and for the establishment of any mutation detection program for this disorder.

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by a severe reduction of visual acuity, hypopigmentation of the retina and the presence of macromelanosomes in the skin and eyes. Various types of mutation have been identified within the OA1 gene in patients with the disorder, including several missense mutations of unknown functional significance. In order to shed light into the molecular pathogenesis of ocular albinism and possibly define critical functional domains within the OA1 protein, we characterized 19 independent missense mutations with respect to processing and subcellular distribution on expression in COS-7 cells. Our analysis indicates the presence of at least two distinct biochemical defects associated with the different missense mutations. Eleven of the nineteen OA1 mutants (approximately 60%) were retained in the endoplasmic reticulum, showing defecNStive intracellular transport and glycosylation, consistent with protein misfolding. The remaining eight of the nineteen OA1 mutants (approximately 40%) displayed sorting and processing behaviours indistinguishable from those of the wild-type protein. Consistent with our recent findings that OA1 represents a novel type of intracellular G protein-coupled receptor (GPCR), we found that most of these latter mutations cluster within the second and third cytosolic loops, two regions that in canonical GPCRs are known to be critical for their downstream signaling, including G protein-coupling and effector activation. The biochemical analysis of OA1 mutations performed in this study provides important insights into the structure-function relationships of the OA1 protein and implies protein misfolding as a major pathogenic mechanism in OA1.

Ocular albinism type I (OA1) is an X-linked disorder characterized by severe reduction of visual acuity, strabismus, photophobia and nystagmus. Ophthalmologic examination reveals hypopigmentation of the retina, foveal hypoplasia and iris translucency. Microscopic examination of both retinal pigment epithelium (RPE) and skin melanocytes shows the presence of large pigment granules called giant melanosomes or macromelanosomes. In this study, we have generated and characterized Oa1-deficient mice by gene targeting (KO). The KO males are viable, fertile and phenotypically indistinguishable from the wild-type littermates. Ophthalmologic examination shows hypopigmentation of the ocular fundus in mutant animals compared with wild-type. Analysis of the retinofugal pathway reveals a reduction in the size of the uncrossed pathway, demonstrating a misrouting of the optic fibres at the chiasm, as observed in OA1 patients. Microscopic examination of the RPE shows the presence of giant melanosomes comparable with those described in OA1 patients. Ultrastructural analysis of the RPE cells, suggests that the giant melanosomes may form by abnormal growth of single melanosomes, rather than the fusion of several, shedding light on the pathogenesis of ocular albinism.

G protein-coupled receptors (GPCRs) participate in the most common signal transduction system at the plasma membrane. The wide distribution of heterotrimeric G proteins in the internal membranes suggests that a similar signalling mechanism might also be used at intracellular locations. We provide here structural evidence that the protein product of the ocular albinism type 1 gene (OA1), a pigment cell-specific integral membrane glycoprotein, represents a novel member of the GPCR superfamily and demonstrate that it binds heterotrimeric G proteins. Moreover, we show that OA1 is not found at the plasma membrane, being instead targeted to specialized intracellular organelles, the melanosomes. Our data suggest that OA1 represents the first example of an exclusively intracellular GPCR and support the hypothesis that GPCR-mediated signal transduction systems also operate at the internal membranes in mammalian cells.

Objective: To report the transmission of unsuspected X-linked ocular albinism in an oocyte donor program., Design: Case report., Setting: University medical center., Patient(s): A 24-year-old white female oocyte donor and the outcomes of three recipient pregnancies., Intervention(s): Clinical assessment and molecular diagnostic tests., Main Outcome Measure(s): Mutation detection., Result(s): Demonstration of carrier status and multiple transmissions of a mutant allele., Conclusion(s): We describe the transmission of a mutant allele for X-linked ocular albinism from an unsuspected carrier female oocyte donor to three independent pregnancies. We emphasize the need for diligent inquisition to clarify any unusual history of ocular or constitutional signs that might signify an X-linked disorder.

Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.

The locus for ocular albinism type 1 (OA1) has been assigned to the Xp22.3 region through both linkage and deletion mapping. The disorder was found to be genetically homogeneous, as all informative families showed convincing linkage data with markers on Xp22.3 and all identified deletions involved in the same region. The OA1 gene recently was cloned and several intragenic deletions were identified in affected individuals. We have characterized the genomic structure of the OA1 gene, which spans approximately 40 kb of genomic DNA and contains nine exons. A highly polymorphic dinucleotide repeat was identified in intron 1, that provides a useful tool for molecular diagnosis. Knowledge of the intron/exon boundaries allowed us to search for point mutations in patients' genomic DNA. All nine exons of the OA1 gene, as well as the 5' and 3' untranslated regions, were scanned for point mutations in PCR-amplified DNA from 60 OA1 patients. The mutations identified include: two frameshifts and a splice site mutation leading to truncated OA1 proteins; a deletion of a threonine codon at position 290; and four missense mutations,two of which involve amino acids located within putative transmembrane domains. Two of the mutations each occur in three apparently unrelated families, consistent with previous observations of a founder effect in OA1. Surprisingly, mutations were detected in only one-third of the patients (21 of 60) ascertained. We postulate that mutations not yet identified in either regulatory elements of the OA1 gene, or in other gene(s) located within the same chromosomal region, may be common cause of X-linked ocular albinism.

Ocular albinism type 1 (OA1) is an X-linked recessive disorder characterized by a major impairment of visual acuity, nystagmus, strabismus, photophobia and retinal hypopigmentation. From the analysis of patients carrying deletions and translocations involving the distal short arm of the X chromosome (Xp22.3) we have identified a region of approximately 110 kb in which the OA1 gene must lie. We have extensively searched for genes in this region using a variety of techniques which included exon amplification, cDNA selection and direct hybridization of cosmid inserts to cDNA libraries. Putative exons identified by exon amplification were used to screen a human retina cDNA library and several cDNA clones corresponding to an approximately 7.5 kb transcript were isolated and characterized. Transcripts of this newly identified gene were found to be abundant in retina and melanoma and could also be detected in brain, placenta, lung, kidney and pancreas. Interestingly, sequence analysis revealed that this new gene encodes a 1616 amino acid protein sharing significant similarities with the Apical Protein from Xenopus laevis (APX) which is implicated in amiloride-sensitive sodium channel activity. The gene, termed APXL (APX-Like), spans approximately 160 kb, contains 10 exons and covers over 70% of the 110 kb critical region for OA1. A truncated pseudogene sharing very high levels of homology with the rat eIF-5 gene, a eukaryotic translation initiation factor, was found to lie in the middle of intron 1. APXL was found deleted in two patients with contiguous gene syndromes including OA1 and in one patient with isolated OA1. Mapping, expression and patient analysis data led us to consider the APXL gene a strong candidate for the OA1 gene. DNA from 57 unrelated patients with OA1 was, therefore, scanned for mutations in the coding region, using both SSCP analysis and direct sequencing. No functionally significant mutation was identified, suggesting that APXL is not directly involved in OA1. Further studies are needed to clarify the physiologic role of this highly conserved gene.

We have applied a yeast artificial chromosome (YAC)-based cosmid isolation and binning strategy to convert a YAC contig in Xp22 into 1.6 Mb of overlapping cosmids. This strategy is based on the screening of a high-density arrayed X chromosome-specific cosmid library with large YAC-derived restriction fragments and entire YAC probes. Cosmids selected in this way were gridded on dot blots and further mapped into bins defined by the overlap intervals of the YACs and YAC fragments. This rapid binning of cosmids simplified the subsequent assembly of cosmid contigs by restriction fingerprint hybridization. In total, we identified 139 cosmids spanning the entire 1.6 Mb region with a minimal overlap set of 53 clones. These cosmids were assigned to 17 bins and 9 contigs. One of the contigs is 665 kb in length and is one of the largest uninterrupted cosmid contigs in humans reported to date. The gaps between the contigs are minor and, together, they represent less than 7% of the region covered. Two previously identified genes are contained in these cosmids, the gene for amelogenin (AMG) and the recently isolated putative chloride channel gene CICN4. In addition, two disease loci have been mapped to this region: X-linked ocular albinism type 1 (OA1) and the microphthalmia with linear skin defects (MLS) syndrome. The assembly of the cosmid maps allowed us to determine the size of the deletion intervals for these two loci, which were estimated to be 110 kb for OA1 and 570 kb for MLS.(ABSTRACT TRUNCATED AT 250 WORDS)

We have compared the efficiency of direct gene transfer in normal and regenerating rat skeletal muscle. Muscle necrosis and regeneration was induced by intramuscular injection of bupivacaine in the soleus muscle of adult rats. Plasmids containing beta-galactosidase (beta-gal) or chloramphenicol acetyltransferase (CAT) genes driven by viral promoters were injected 3 days after bupivacaine treatment into the regenerating and the contralateral uninjured muscles. Expression of CAT activity was > 80-fold higher in regenerating compared to control muscles at 7 days post-transfection, but decreased at 30 and 60 days. Southern blot analysis showed that the predominant form of CAT DNA was episomal in transfected muscles; however, CAT activity measurements performed on the same transfected muscles showed no precise correlation between enzymatic activity and amount of plasmid DNA. Expression of beta-gal was detected in numerous regenerating fibers of the injured soleus muscles at 7 days post-transfection; in contrast, only rare positive fibers were found in control muscles. Focal infiltrates of mononuclear cells, which surround and invade selectively beta-gal-positive fiber segments, were observed at 30 days post-transfection, suggesting that immune mechanisms are implicated in the progressive loss of transgenes with time. The finding that regenerating muscle fibers display a higher efficiency of transfection may be relevant to gene therapy of Duchenne muscular dystrophy, because regenerating fibers are numerous in the early stages of the disease.