Melanosomes – dark organelles enlighten endosomal membrane transport

Introduction

The endosomal system comprises a complex network of organelles and membrane sub-domains with important functions in signal transduction, nutrient uptake, pathogen destruction and/or propagation, and other essential processes^1,2. Although great advances have been made in defining basic mechanisms that underlie membrane dynamics in the secretory pathway, the endosomal system has been more difficult to functionally dissect due to redundancy and compensation by alternative routes. Moreover, the mammalian endocytic network is more complex relative to that of simple eukaryotes such as the yeast Saccharomyces cerevisiae; thus, it is often difficult to infer specific molecular functions in mammals based on the phenotype of mutant yeast strains.

Recent work on the melanosome has provided insights into mammalian endosomal membrane dynamics by revealing how specific trafficking events are exploited to generate tissue-specific organelles. Melanosomes are intracellular organelles that are uniquely generated by pigment cells in the skin and eye, where they function to synthesize and store melanin pigments. Although melanosomes are considered to be lysosome-related organelles (LRO), recent data show that their contents derive from early endosomal membranes. Many new insights have emerged from hereditary diseases in humans and model organisms characterized by reduced or absent pigmentation, often combined with additional seemingly unrelated disorders such as bleeding diathesis, lung fibrosis, and immunodeficiency, due to generalized LRO defects³. Identification of the genes that are mutated in these diseases and functional characterization of their products has dramatically advanced our understanding of pigment cell biology and of the molecular features that underlie melanosome biogenesis. Many of these features have parallels in LROs in vertebrates and primitive eukaryotes, such as the eye pigment granules of Drosophila melanogaster and the gut granules of Caenorhabditis elegans.

This review highlights recent insights into melanosome biogenesis that have advanced our general understanding of organelle formation and endosomal protein sorting. We also discuss important connections between melanosome morphogenesis, neurodegenerative disorders and general mechanisms of endosomal partitioning. Last, we propose models to explain endosomal membrane dynamics required for the formation of conventional and specialized organelles.

Melanosome composition and function

Melanins are complex pigments that provide the skin, hair and eyes of mammals with colour and photoprotection against ionizing radiation. They also function in the development of the optic nervous system and regulate retinal function⁴. Melanins typically consist of copolymers of black and brown eumelanins and red and yellow pheomelanins⁵. Melanin synthesis in epidermal and ocular melanocytes and in pigment epithelia of the retina, iris and ciliary body of the eye occurs within melanosomes^6,7. Sequestration within melanosomes protects components of the cytosol and other membranous organelles from oxidative attack during melanin synthesis, and concentrates melanins for storage (in eye pigment cells) or cell transfer (from epidermal melanocytes to keratinocytes in the skin and hair). Melanosomes that harbour primarily pheomelanins differ in structure and composition from melanosomes with primarily eumelanins⁸, are much less well characterized, and will not be discussed further.

The formation of premelanosome fibrils

PMEL is the main constituent of the intralumenal proteinaceous fibrils of stage I and II premelanosomes (FIG. 2). The fibrils are immunoreactive for PMEL by microscopy and biochemical analyses²⁴, and expression of PMEL alone in non-melanocytic cells²² or even in vitro²⁵ results in the formation of morphologically similar fibrils (although PMEL fibrillogenesis may be modified by other melanocyte-specific proteins such as MART1²⁶ through as yet unknown mechanisms). By contrast, melanosomes from silver mice — in which PMEL is depleted due to a truncation of the cytoplasmic domain (see below) — lack fibrillar structure and are consequently enlarged and rounded²⁷. Thus, PMEL is both necessary and sufficient for premelanosome fibril formation and melanosome shape. The importance of the fibrils in pigment-cell function is supported by pigmentation defects — most likely due to loss of melanocyte viability^28,29 — in Pmel-mutant zebrafish³⁰, dogs³¹, horses³², chickens and mice²⁴.

Mature fibrils copurify with fragments of the PMEL lumenal domain but not with full-length PMEL^14,33. The largest of these fragments, known as Mα, encompasses the N-terminal 443 lumenal residues of the largest human splice isoform and rapidly forms fibrils in vitro²⁵. Mα is proteolytically cleaved from the remaining PMEL fragment (Mβ) in a post-Golgi compartment, likely endosomes³⁴, by a proprotein convertase³³ (FIG. 2). Inhibition of proprotein convertase activity in melanocytes or elimination of the PMEL cleavage site in transfected HeLa cells blocks the formation of fibrils and of morphologically intact melanosomes³³, which indicates that cleavage is required for fibrillogenesis. Similarly, proprotein convertase cleavage of von Willebrand factor — a coagulation regulatory protein— underlies the formation of a distinct type of tubular fibril in Weibel-Palade bodies³⁵.

PMEL sorting within MVBs en route to premelanosomes

PMEL is synthesized by melanocytes, and thus must navigate the secretory pathway to accumulate in premelanosomes. Nevertheless, unlike the tubular von Willebrand factor-containing fibrils of Weibel-Palade bodies that begin to form in the trans-Golgi network (TGN) of endothelial cells⁴⁰, PMEL-containing fibrils are not observed in early secretory compartments of melanocytes. How does PMEL access premelanosomes in a pre-fibrillar form? Although a contrasting view of PMEL trafficking and premelanosome formation has been invoked based on immunoreactivity in isolated subcellular fractions^14,41, a wealth of evidence supports the notion that endosomes are an obligate intermediate in PMEL processing²⁴.

HPS and sorting of melanosomal enzymes

Mature (stage III and IV) melanosomes are characterized by pigment deposits over the fibrils, and unlike stage II melanosomes, harbour melanogenic enzymes like tyrosinase and TYRP1. These proteins must be delivered to preformed premelanosomes such that melanin synthesis begins only after the complete formation of the fibrils on which melanin subsequently accumulates. This implies that melanogenic enzymes are delivered by pathways distinct from those used by premelanosome components such as PMEL. Several observations initially suggested that melanogenic enzymes could be transferred directly from the TGN to melanosomes by clathrin-coated vesicles^13,49–51, a model supported by the finding that tyrosinase and TYRP1 accumulate in the pericentriolar "TGN" area in hypopigmented melanocytes that fail to synthesize glycosphingolipids⁵². However, the existence of a pericentriolar pool of early endosomes², often bearing clathrin-coated buds, challenges some assumptions of the TGN-to-melanosome model. Recent studies, exploiting genetic deficiencies in humans and mouse models that affect pigmentation at late stages of melanogenesis, now support the notion that tyrosinase and TYRP1 are preferentially sorted to melanosomes from early endosomes.

Hermansky-Pudlak Syndrome (HPS) is a multisystem disorder characterized by partial albinism, excessive bleeding, and often lung fibrosis, sometimes accompanied by immunodeficiency and/or granulomatous colitis²³. These systemic symptoms result from the malformation or malfunction of LRO, including melanosomes, platelet dense granules, and in some cases lung lamellar bodies and/or cytotoxic T-cell granules. HPS results from mutations in any of at least 8 genes; a similar disorder in mice, first identified based on coat colour dilution, results from mutations in at least 15 different genes, including the orthologues of the genes mutated in human HPS^23,53. Although the HPS-associated genes are expressed in many cell types, they appear to be functionally essential for the generation of a restricted number of LROs within affected cell types. Most of the genes encode subunits of multi-subunit protein complexes (BOX 3). Some of the complexes, such as AP-3, are known to be involved in vesicular transport, whereas others, such as the biogenesis of lysosome-related organelle complex-1 (BLOC-1), BLOC-2 and BLOC-3, consist of subunits that lack common structural motifs or homology to proteins of known function (BOX 3). Biochemical analyses of these proteins and functional analyses of melanocytes from HPS models are shedding light on melanosomal enzyme trafficking and on the function of AP-3 and BLOCs in regulating early endosomal dynamics.

AP complexes in melanosome protein sorting

Mutations in subunits of the AP-3 complex have been identified in HPS type 2 patients, pearl and mocha mice, and D. melanogaster and C. elegans mutants with eye pigment and gut granule defects, respectively^54,55. AP-3 is one of four heterotetrameric adaptor complexes that function in linking transmembrane cargo, through cytoplasmic sorting signals, to coated bud formation on post-Golgi membranes⁵⁶. In melanocytes, AP-3 function has best been described with respect to its role in tyrosinase trafficking. The cytoplasmic domain of tyrosinase harbours a di-leucine-based sorting motif^15,57 that binds to AP-3 in vitro and is required for sorting to lysosomes or neurosecretory granules in other cell types^58–60. Importantly, a cohort of tyrosinase is mislocalized to endosomes in melanocytes from HPS type 2 patients and pearl mice^15,61. As AP-3 coats are found primarily on buds on early endosomal tubules^15,62 (FIG. 3A) that also contain tyrosinase in normal melanocytes¹⁵, AP-3 likely functions in tyrosinase sorting from endosomes. The mislocalized tyrosinase in AP-3-deficient cells accumulates primarily in early endosomes and in intralumenal vesicles of MVBs¹⁵, which suggests that AP-3 may have a function in vertebrate cells that is similar to its function in yeast⁶³, to divert cargo from the MVB pathway.

Although a cohort of tyrosinase is mislocalized to endosomes and pigmentation is attenuated in pearl and HPS type 2 melanocytes, these cells harbour pigmented mature melanosomes that are almost indistinguishable from the melanosomes in control melanocytes and retain a cohort of active tyrosinase^15,64,65. This finding indicates that an additional pathway that is independent of AP-3 must deliver tyrosinase to melanosomes. Indeed, the tyrosinase cytoplasmic di-leucine-based sorting signal also binds in vitro to a second heterotetrameric adaptor complex, AP-1¹⁵. Moreover, tyrosinase is found at steady state in distinct endosomal buds that contain either AP-3 or AP-1 in wild-type melanocytes, and the cohort within AP-1-containing buds increases in AP-3-deficient cells¹⁵. The presence of distinct AP-1- and AP-3-coated endosomal buds (FIG. 3A, C) is not unique to melanocytes, but is also found in other cell types⁶², in which AP-1 likely functions in recycling mannose 6-phosphate receptors to the TGN⁶⁶ and other receptors to the cell surface⁶⁷.

Whether AP-3 and AP-1 mediate redundant tyrosinase sorting pathways from endosomes to melanosomes is not clear, but a distinct role for AP-1 in melanosomal sorting is suggested by analysis of another mature melanosome cargo protein, TYRP1. TYRP1 localization to melanosomes is not grossly affected by AP-3 deficiency^61,68. Moreover, TYRP1 harbours a di-leucine-based sorting signal²¹ that interacts with AP-1 but not AP-3¹⁵, and colocalizes extensively with AP-1 (but not AP-3) in melanocytic cells¹³ (FIG. 3C). AP-1 is therefore likely to function in TYRP1 transport to melanosomes, and may “rescue” a cohort of tyrosinase that fails to be sorted by AP-3. How these two adaptors cooperate sequentially or concomitantly remains unknown.

Regulation of sorting by BLOCs

Cargo sorted by AP-3 is destined for late endosomes and lysosomes in several cell types; however, in pigment cells, tyrosinase and TYRP1 are destined for melanosomes, which indicates that additional effectors must participate in their sorting. Some of these effectors include BLOCs and other complexes that are defective in HPS.

HPS-associated proteins in other trafficking functions

Functions of other melanosomal cargo

The defects in melanosome biogenesis that are observed in HPS and related models cannot be explained by effects on delivery of solely melanogenic enzymes. For example, because tyrosinase expression alone is sufficient to produce melanin in fibroblasts²⁰, the severe hypopigmentation in BLOC-1-deficient melanocytes belies the cohort of tyrosinase delivered to melanosome-like compartments in these cells⁶⁸. Indeed, additional melanocyte-specific constituents influence melanosome biogenesis (BOX 1). Analogous proteins likely function in the generation and maintenance of other LRO and perhaps all endosomal compartments.

Membrane transporters comprise a major class of proteins that regulate melanosome formation. Mutations in two transporters — OCA2/P and SLC45a2 (also known as AIM1 or MATP) — are respectively responsible for oculocutaneous albinism (OCA) types 2 and 4 in humans and corresponding mouse models^99,100. In OCA2/P-deficient melanocytes, melanosome architecture is severely altered and tyrosinase and TYRP1 are mislocalized^99,101, which suggests a role for OCA2/P in regulating delivery of enzymes to maturing melanosomes. OCA2/P is homologous to bacterial arsenate transporters⁹⁹ and regulates sensitivity to arsenic toxicity¹⁰², which suggests that OCA2/P may function as an anion transporter, perhaps providing counterions for proton transport across melanosome membranes and intralumenal pH regulation^99,103. OCA2/P-deficient melanocytes also have early secretory pathway defects¹⁰⁴, but it is not clear whether these defects reflect OCA2 function in early secretory events or an indirect effect of cellular ion imbalance.

SLC45a2 has been identified in melanosome subcellular fractions¹⁷ and bears homology to sucrose/proton symporters in plants¹⁰⁰, so it might regulate osmoregulation within melanosomes. SLC45a2-deficient melanocytes show abnormalities in melanogenic enzyme trafficking and loss of mature melanosomes¹⁰⁵. A third putative membrane transporter, SLC24a5, was recently identified by positional cloning of the gene responsible for the golden mutation in zebrafish, which affects melanosome size, shape and number in the choroid¹⁰⁶. SLC24a5 is a member of a family of potassium-dependent sodium/calcium exchangers, localizes to vesicular compartments in human melanocytes¹⁰⁶, and copurifies with melanosomes¹⁷. Inhibitor and gene profiling analyses suggest that melanosomal sodium/hydrogen exchangers participate in pigmentation¹⁰⁷, and additional solute transporters have been identified in melanosome subcellular fractions by proteomic analysis¹⁷.

Full melanosome function requires two other classes of protein. Genetic deficiency of OA1, a 7-transmembrane domain protein homologous to G-protein coupled receptors, is responsible for ocular albinism type I¹⁰⁸. OA1-deficient RPE and choroidal melanocytes, and to a lesser extent skin melanocytes, have fewer, enlarged melanosomes relative to controls¹⁰⁸. OA1 is localized to mature melanosomes and late endosomes and can signal through associated Giα proteins^108,109, but neither the natural OA1 ligand nor how signalling might regulate melanosome size is known. A second unexpected contributor to melanosome biogenesis is presenilin, a component of the γ-secretase complex that is responsible for intramembrane cleavage of the Alzheimer’s precursor protein and Notch¹¹⁰. Selective ablation of presenilin expression or function in melanocytes results in hypopigmentation due to impaired transport of tyrosinase and perhaps other proteins to melanosomes, coupled with unusual proteolytic processing of tyrosinase, TYRP1 and DCT¹¹¹. It is possible that presenilin regulates accessibility of membrane proteins that are required for melanogenic enzyme sorting to or fusion with melanosomes.

Conclusions and perspectives

The emerging picture (FIG. 4) suggests that melanosomes form by sequential, timed delivery of cargo from distinct endosomal domains, permitting progressive organelle maturation. The vacuolar domain of early endosomes provides cargo — including PMEL — required for the underlying melanosome structure, and is the likely source of the stage II melanosome limiting membrane. Distinct tubular domains of early endosomes then provide additional cargo — including melanogenic enzymes — that generate melanins and mediate organelle maturation through stages III and IV. The main challenges for the future will be to understand how these different targeting pathways are coordinated, how the molecular machinery functions in segregating these distinct domains, how tissue-specific membrane proteins shape the targeting pathways, and how ubiquitous protein complexes — like those deficient in HPS — are co-opted for the specific use of generating a distinct lineage of LRO.

One major unresolved question is how cargoes are segregated within stage I melanosomes for the conventional late endocytic pathway or stage II melanosomes – a process that does not occur in “non-specialized” cell types. Does Pmel trafficking, particularly ESCRT-independent sorting in MVBs, generate a “novel” endosomal intermediate within melanocytes distinct from late endosome precursors? Or do the fibrils themselves promote segregation from the intralumenal membranes from which they derive, separating the organelle into distinct domains – a fibrillar region and a multivesicular region – that eventually segregate toward two fates? Intriguingly, preliminary data suggest that as the PMEL-containing MVBs mature to stage II premelanosomes, they lose most of their internal membrane vesicles and either recruit or unmask effectors that render them competent to undergo further maturation to stage III and IV by docking and fusing with transport intermediates that carry melanogenic enzymes. These data might favour a model either in which the internal membranes segregate to a distinct domain or back-fuse to the limiting membrane, as occurs in antigen-processing compartments during dendritic-cell maturation¹¹².

It is surprising that none of the HPS models analysed thus far interfere with either the segregation of stage II melanosomes from endosomes or grossly with fibril formation. This parallels the minimal effects of several HPS models on the formation of α granules in platelets — which, like stage II melanosomes, develop from MVBs¹¹³ — despite the complete loss of dense granules¹¹⁴. The underlying mechanisms behind these maturation events may be essential for normal endosomal maturation, such that mutations that affect them are not tolerated.

The lumenal domain-dependent mechanism of PMEL invagination onto internal membranes and the role of the internal membranes in regulating fibrillogenesis likely represent important ubiquitous events and must be better defined. Increasing evidence supports the role of MVBs and their associated lipids or derived exosomes in the formation of amyloid in Alzheimer’s and the prion diseases^115–117. The fast rate and high efficiency of PMEL fibrillogenesis in vitro²⁵ may provide a better mechanistic understanding of the role of such lipids in general processes of amyloidogenesis.

Another major unresolved question is how ubiquitous protein complexes, such as AP-3, the BLOCs, and HOPS, create distinct functional endosomal domains that function in tissue-specific sorting events. Perhaps each complex functions in concert with as-yet undefined tissue-specific factors, such as RAB32 or RAB38, to assemble specific macromolecular complexes at defined sites on endosomal membranes. Alternatively or additionally, altered expression levels of ubiquitous components might shift reaction equilibria in such a way as to generate novel pathways. For example, the increased expression of endosomal SNARE proteins in melanocytic cells⁷⁸ might favour formation of novel trans-SNARE complexes or divert associated BLOCs or HOPS from “constitutive” pathways. Defining the SNARE distribution among melanosomal and endosomal membranes and the complexes they form in melanocytes will be critical for testing these hypotheses. The molecular functions of the BLOCs and HOPS complex must also be defined. How do they functionally interact with coats and SNAREs? How does BLOC-1 facilitate sorting of TYRP1 and other cargo toward melanosomes, and does it act in concert with adaptor complexes? To address these questions, it will be necessary to further define the subcompartmentalization of the endosomal system in these highly specialized cells and the protein-protein interactions that mediate it.

Finally, we need to learn more about the lumenal milieu of melanosomes and related organelles, and how the establishment of solute gradients regulates protein sorting. Transporters are becoming increasingly recognized as key regulators of membrane dynamics in the endocytic pathway (for example in cholesterol metabolism¹¹⁸). The characterization of melanosome membrane transporters and the availability of cell lines in which they fail to function provide an unprecedented opportunity to dissect how transporters modulate membrane dynamics via the lumenal environment of source and target compartments.

Our advances in melanosome biogenesis will allow us to better understand how other LRO form and the subcompartmentalization of the endosomal pathway in specific cell types. For example, endothelial cells use similar pathways to generate Weibel-Palade bodies, endosome-granule interactions may be required for maturation of cytolytic granules in T cells, and platelets likely use HPS-encoded protein complexes in similar ways to generate dense granules¹⁹. Thus, the dark organelle is clearly shedding light on the future of endosomal trafficking and the formation of specialized organelles.

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