Browsing by Author "Di Pietro, Santiago, advisor"
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Item Open Access Cargo induced recruitment of the endocytic adaptor Sla1 and the role of Sla1-clathrin binding in endocytosis(Colorado State University. Libraries, 2018) Tolsma, Thomas O., author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Reist, Noreen, committee memberClathrin-mediated endocytosis is a highly dynamic process that is essential in all eukaryotes. This process is utilized for a number of functions including the uptake of extracellular nutrients, manipulation of the plasma membrane content, downregulation of cell signaling pathways, and viral entry. While differences in protein composition, sequence, and structure do exist between species for this process, many core protein functions and the mechanistic steps involved in endocytic vesicle formation and internalization are highly conserved. This has allowed findings from one species to be applicable to another. For this reason Saccharomyces cerevisiae has been characterized as a highly useful model organism for studying and identifying key proteins and conserved mechanisms in clathrin-mediated endocytosis that are found in all eukaryotes. In yeast, roughly 60 proteins have been identified as being part of the endocytic machinery. Clathrin-mediated endocytosis begins with the recruitment of early endocytic proteins that establish the site of endocytosis. This includes scaffolding and coat proteins, such as clathrin, that aggregate at the plasma membrane through interactions with lipids, protein cargo, and other components of the endocytic machinery. This is followed by recruitment of other late coat proteins that further prepare the site for internalization. Following coat formation the mobile phase of membrane invagination is initiated by the recruitment of the actin polymerization machinery. Actin polymerization then generates an inward force at the site of endocytosis that causes invagination of the plasma membrane. The invagination is then separated from the plasma membrane through the recruitment of scission proteins that pinch off the endocytic vesicle. Lastly the internalized vesicle undergoes a process of coat protein disassembly before being targeted to its proper destination in the cell. While much of this process has been well characterized, significant gaps in our understanding of how different steps in endocytic progression are coordinated and how endocytic proteins function still exist. Using a combination of yeast genetics, fluorescent microscopy, electron microscopy, and biochemistry we have furthered our understanding of clathrin-mediated endocytosis, focusing on the role adaptor-clathrin and adaptor-cargo binding plays in formation and progression of the endocytic process. Our work began by focusing on the role of the adaptor protein Sla1, a clathrin and cargo binding protein that serves essential roles in endocytosis. It was previously established that Sla1 binds clathrin through a variable clathrin box of sequence LLDLQ. Loss of clathrin binding by mutation of this clathrin box has a dramatic effect on endocytosis such as an increased patch lifetime of Sla1 at endocytic sites, and dramatic defects in internalization of endocytic protein cargo. While these experiments demonstrated the importance of Sla1-clathrin binding in endocytosis, they did not explain why Sla1-clathrin binding was important and how this interaction contributes mechanistically to endocytic progression. By imaging Sla1 and clathrin, our work demonstrates that Sla1 contributes to proper clathrin recruitment to endocytic sites. A loss of proper recruitment of clathrin to endocytic sites by mutation of the Sla1 variable clathrin box also resulted in significant accumulation of other endocytic proteins, including those involved in actin polymerization. The lifetime of these additional endocytic components lasted for significantly longer at endocytic sites, some having a disruption in normal recruitment dynamics. Despite this accumulation of the actin polymerization machinery, there is a significant delay in actin polymerization and an increase in actin polymerization time and levels at endocytic sites. Our results also demonstrate defects in the formation of the endocytic invagination and delays in scission. Thus, the Sla1-clathrin interaction is needed for normal progression through different stages of the endocytic process. A second question in the endocytic field that has received little attention is the role cargo plays in the recruitment of the endocytic machinery. The conventional view is that first the endocytic machinery forms an endocytic site and then cargo is concentrated by binding adaptor proteins. Sla1 has previously been shown to bind to endocytic protein cargo that contains the amino acid sequence NPFxD through its SHD1 domain. It has also been shown through biochemical experiments that Sla1 binds Ubiquitin via its third SH3 domain. Both NPFxD and Ubiquitin have been shown to be important signals of cargo for entry into the endocytic pathway. The question, however, remained as to whether cargo binding via these signals contributes to recruitment of the adaptor Sla1 to endocytic sites. The work described in this dissertation will present evidence that this is indeed the case.Item Open Access Determination of the functions of Rab32, Rab38, and their effector Myosin Vc in the biogenesis of melanosomes(Colorado State University. Libraries, 2013) Bultema, Jarred, author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Chen, Chaoping, committee member; Reddy, Anireddy, committee memberIn mammals, pigment produced within specialized cells is responsible for skin, hair, and eye coloration. Melanocytes are specialized cells that produce pigment within an organelle known as the melanosome. Melanosomes are a member of a specialized class of organelles, known as Lysosome-related organelles (LRO), which are responsible for a number of critical functions in mammals such as pigmentation, blood clotting, lung function, and immune function. LROs are related to the ubiquitous lysosome, and are formed using the same molecular mechanisms as lysosomes that rely upon the Adaptor Protein complexes -1 (AP-1) and -3 (AP-3), and the Biogenesis of Lysosome-related Organelles Complex (BLOC)-2 (BLOC-2). These protein complexes are critical for the trafficking of specialized cargoes to melanosomes required for proper melanin synthesis. But, these complexes are also used for the formation of lysosomes, and no mechanism is known to distinguish between trafficking to lysosomes and melanosomes. The melanosome serves as a model system to study the formation of LROs, and insights from the study of melanosomes help explain the biogenesis of other LROs. In this dissertation, I present the finding that Rab32 and Rab38 function as melanosome-specific trafficking factors that allow for the use of AP-3, AP-1, and BLOC-2 in melanosome biogenesis. Using biochemical approaches, I show that Rab32 and Rab38 bind directly to AP-3, AP-1, and BLOC-2 on membranes. In microscopy experiments, I demonstrate that Rab32 and Rab38 localize to early endosomal subdomains where AP-3, AP-1, and BLOC-2 function. Using a combination of biochemical and microscopic approaches, I show that Rab32 and Rab38 serve partially redundant functions in trafficking of specialized cargoes to melanosomes. I report the discovery that Myosin Vc, a class V myosin motor, interacts with Rab32 and Rab38 and serves novel functions in melanosomes trafficking. I show, using biochemical approaches, that Myosin Vc directly binds to several melanosomal Rab proteins and serves as an effector of these proteins in melanosome biogenesis. Using a combination of approaches, I demonstrate that depletion of Myosin Vc from melanocyte cells causes defects in the trafficking of cargoes to melanosomes, but also causes severe defects in the secretion of mature melanosomes. With biochemical and microscopic approaches, I compare the function and localization of Myosin Vc in melanocytes to related proteins Myosin Va and Myosin Vb, and provide evidence to suggest that all three of these proteins function in distinct steps of melanosome trafficking. My results answer outstanding questions about the use of ubiquitous trafficking machinery (AP-3, AP-1, and BLOC-2) in trafficking to a specialized organelle. I provide evidence to answer outstanding questions about the mechanism of action of Rab32 and Rab38 in melanosome trafficking through my studies with Myosin Vc. I also establish new areas of research in the comparison of Myosin Va, Myosin Vb, and Myosin Vc in melanosome trafficking. My results address numerous unknown areas in melanosome biogenesis, expand the knowledge of melanosome biogenesis, and provide numerous new avenues of research to explore to understand specialized trafficking to LROs.Item Open Access Discovery and characterization of the SLAC complex and its role in actin polymerization during clathrin-mediated endocytosis(Colorado State University. Libraries, 2013) Feliciano, Daniel, author; Di Pietro, Santiago, advisor; Bamburg, James, committee member; Curthoys, Norman, committee member; Chen, Chaoping, committee member; Reist, Noreen, committee memberEndocytosis is the process by which cells control the lipid and transmembrane protein compositions in order to comply with certain requirements essential for cellular function. The different forms of endocytosis provide the cell with a discriminatory system where specific cargoes are selected, packed and internalized when there is a particular physiological demand. Given the importance of endocytic internalization routes for a variety of cellular processes, it is not surprising that defects in the protein machinery involved in these pathways leads to pathologies. Examples of some metabolic disorders associated to defects in adaptor or receptor function include autosomal recessive and familial hypercholesterolemia. In other cases, mutations in actin regulatory proteins, such as WASp, can cause many blood disorders that include primary immunodeficiency and thrombocytopenia. Clathrin-mediated endocytosis (CME) is a fundamental pathway conserved from yeast to humans that proceeds by forming a clathrin coat at the plasma membrane followed by the recruitment of proteins that promote membrane curvature, actin polymerization, and scission. CME is the mayor route for nutrient uptake, distribution of membrane components, and receptor internalization. During CME, branched actin polymerization nucleated by the Arp2/3 complex provides force needed to drive vesicle internalization. Las17 (WASp) is the strongest activator of the Arp2/3 complex in yeast cells, it is not autoinhibited, and arrives to endocytic sites 20 seconds before actin polymerization begins. One of the most outstanding questions in the field has been how Las17 is inhibited during the initial 20 seconds after its arrival to sites of endocytosis. In this dissertation, the discovery and characterization of a stable complex between Las17 and the clathrin adaptor Sla1 is described, in which Las17 is inhibited. This interaction is direct, multivalent, and strong, and was mapped to novel Las17 polyproline motifs that are simultaneously class I (RxxPxxP) and class II (PxxPxR). In vitro pyrene-actin polymerization assays established that Sla1 inhibition of Las17 activity depends on a new class I/II Las17 polyproline motifs. The inhibition is based on competition between Sla1 and monomeric actin for binding to sequences comprising a novel G-actin binding site in Las17 that is also characterized. The Las17 novel G-actin binding module 1 (LGM1) requires two sets of arginine-rich sites for normal Las17 function in vitro and in vivo. Furthermore, live cell imaging showed the interaction with Sla1 is important for normal Las17 recruitment to endocytic sites, its inhibition during the initial 20 seconds, and for efficient endocytosis. Within this complex, Las17 requires full length Bzz1, a membrane tubulation protein, for its activation in vitro through a mechanism that does not depend on complex dissociation. Since Sla1 and Las17 regulate actin polymerization during clathrin-mediated endocytosis, this complex has been named SLAC. The discovery and characterization of the SLAC complex help to define the negative and positive mechanisms regulating Las17 activity and answer one of the most outstanding questions in the field. This work also sets the stage to decipher the roles of other WASp homologues in mammalian cells. Overall, findings reported here advance our understanding of the regulation of actin polymerization by Las17 during clathrin-mediated endocytosis.Item Open Access Exploring the roles of Bzz1 in clathrin-mediated endocytosis(Colorado State University. Libraries, 2015) Barry, Lauren Marie, author; Di Pietro, Santiago, advisor; Curthoys, Norm, committee member; Glycenfer, Frances, committee memberClathrin-mediated endocytosis (CME) is a highly conserved process in eukaryotes. This particular route of endocytosis plays an integral role in virus internalization, nutrient uptake, regulation of signal transduction, and overall cell health. CME can be characterized by three distinct stages: coat formation, cargo binds a receptor on the plasma membrane which leads to the recruitment of coat proteins and the formation of a clathrin coated pit; actin polymerization, globular actin will polymerize in such a way that the vesicle can overcome the turgor pressure of the plasma membrane; and scission and coat disassembly, the vesicle is separated from the plasma membrane and the coat proteins are recycled for the next endocytic event. Initiation of actin polymerization is mediated by the activation of the Arp2/3 complex. Arp2/3 is activated by Las17, the yeast homolog of human WASp (Wiskott-Aldrich Syndrome protein). Las17 interacts with multiple proteins, both activators and inhibitors. The activity of Las17 is inhibited by a protein called Sla1. Sla1 binds the P8-12 region of Las17 and blocks one of two globular actin binding sites. Sla1 and Las17 form a stable complex referred to as SLAC (Sla1, Las17, Actin, and Clathrin) and arrive at the plasma membrane together. This project deals specifically with the interaction between the SLAC complex and the putative activator Bzz1 and explores the possibility that Bzz1 both activates actin polymerization and tubulates the plasma membrane. Understanding the mechanisms that allow clathrin-mediated endocytosis to progress from start to finish is critical. Wiskott-Aldrich Syndrome, hypercholesterolemia, and neurodegenerative disorders can all be tied to defects in CME.Item Embargo Identification of the TPC2 interactome reveals TSPAN10 and OCA7 as key players in the biogenesis of melanosomes(Colorado State University. Libraries, 2023) Beyers, Wyatt, author; Di Pietro, Santiago, advisor; Amberg, Gregory, committee member; Santangelo, Thomas, committee member; Yao, Tingting, committee memberMany specialized cell types gain their function through the generation of specialized organelles that make or store cell-specific biomolecules. A group of specialized organelles are called Lysosome Related Organelles (LROs) because they are derived from Golgi and endolysosomal compartments and their biogenesis depends on trafficking pathways and machinery shared with lysosomes, many have protein contents partially overlapping with lysosomes, and typically have low pH during stages of their maturation. One well-studied model LRO is the melanosome, the organelle in melanocytes and retinal pigment epithelial cells responsible for melanin pigment production in the eyes, hair, and skin, and defects in melanosome function lead to pigmentation diseases such as oculocutaneous albinism. Melanosome biogenesis is a complex process requiring ubiquitous membrane trafficking machinery to be repurposed for the differentiation of melanosomes from other endosomal compartments and specific delivery of melanosome synthesizing enzymes, Tyrosinase and Tyrosinase Related Proteins 1 and 2. Furthermore, correct melanosome maturation requires remodeling of the melanosome membrane, recycling of membrane trafficking machinery, generation of intraluminal amyloid fibrils with the correct structure for melanin packaging, tight pH control, as well as coordinated influx of copper, zinc, tyrosine, and cysteine for melanin synthesis. These processes require the temporospatial coordination of at least 100 known proteins, and probably dozens more remain undiscovered. In this dissertation, I present the discovery of new proteins involved in the biogenesis of melanosomes. Proximity biotinylation by promiscuous biotin ligase enzymes followed by biotin pulldown and mass spectrometry has emerged as a powerful technique for the identification of protein-protein interactions, protein complex determination, and identification of organelle membrane proteomes. I utilized the melanosome localized cation channel, TPC2, genetically fused with the BioID2 biotin ligase, to identify proteins in proximity to TPC2 at the cytosolic surface of melanosome membranes of MNT1 melanoma cells. Through mass spectrometry analysis of biotinylated proteins enriched through Streptactin pulldown, a TPC2 proximity interactome was identified comprising over 200 proteins. Subsequent fluorescence confocal microscopy analysis confirmed several proteins, including PLD1, SV2A, TSPAN10, and OCA7/C10orf11/LRMDA all colocalize highly with TPC2-EGFP, confirming they are new melanosome proteins. In follow-up functional studies, TSPAN10 and OCA7 were confirmed to be involved in pigmentation, with severe melanin depletion in TSPAN10 or OCA7 knockout MNT1 cells. TSPAN10 and OCA7 both influence the processing of the PMEL protein, which is required for correct melanosome ultrastructure and for melanin packaging. Further investigation of TSPAN10 revealed it functions with the pigmentation associated metalloproteinase, ADAM10, and is required for ADAM10 expression and localization to endosomal compartments. On the other hand, OCA7 was found to work with the melanosome localized Rab proteins, Rab32 and Rab38, and regulates the pH of melanosomes. Thus, the newly defined TPC2 interactome in melanocytes was proven as a valuable dataset that robustly identifies new melanosome proteins. Chapter 1 of this dissertation provides a broad overview of membrane trafficking pathways, as well as a synopsis of the specific proteins and pathways involved in melanosome biogenesis and homeostasis. Chapter 2 investigates the TPC2 interactome in MNT1 cells, and it characterizes TSPAN10 as a new player in melanosome biogenesis. Finally, Chapter 3 provides a characterization of the OCA7 protein associated with oculocutaneous albinism type 7 and investigates OCA7 function using a newly generated OCA7 knockout cell model.Item Open Access Investigating new protein components of the endocytic machinery in Saccharomyces cerevisiae(Colorado State University. Libraries, 2016) Farrell, Kristen, author; Di Pietro, Santiago, advisor; Bamburg, James, committee member; Krapf, Diego, committee member; Tamkun, Michael, committee memberClathrin-mediated endocytosis is an essential eukaryotic process which allows cells to control membrane lipid and protein content, signaling processes, and uptake of nutrients among other functions. About 60 proteins have been identified that compose the endocytic machinery in Saccharyomes cerevisiae, or budding yeast. Clathrin-mediated endocytosis is highly conserved between yeast and mammals in terms of both protein content and timing of protein arrival. First, there is an immobile phase in which clathrin and other coat components concentrate at endocytic sites. Second, another wave of proteins assembles about 20 seconds before localized actin polymerization. Third, a fast mobile stage of endocytosis occurs coinciding with local actin polymerization and culminates with vesicle scission. Fourth, most coat proteins disassemble from the internalized vesicle. Despite the knowledge of so many endocytic proteins, gaps still remain in the complete understanding of the endocytic process. We attempt to fill some of these gaps with a screen of the yeast GFP library for novel endocytic-related proteins using confocal fluorescence microscopy. We identified proteins colocalizing with RFP-tagged Sla1, a clathrin adaptor that serves as a well-known marker of endocytic sites. Ubx3 and Tda2, two unstudied proteins, were selected for further investigation based on high degree of colocalization with Sla1. Ubx3 shows fluorescent patch dynamics similar to an endocytic coat protein. Ubx3 is dependent on clathrin for patch lifetime and binds clathrin via a W-box, the first identification of this clathrin binding motif in a non-mammalian species. Uptake assays performed in a knockout strain of Ubx3 display a reduction in both bulk endocytosis by fluorescent dye Lucifer Yellow and cargo-specific endocytosis by methionine transporter Mup1. The Ubx3 knockout cells also show a significant increase in lifetime of early endocytic protein Ede1, and removing its UBX domain alone results in similar defects to the Ubx3 knockout. The endocytic defect may be due to lack of recruitment of ubiquitin regulator AAA ATPase Cdc48 to the endocytic site. Inactivation of Cdc48 reduces Lucifer yellow uptake to minimal levels and causes aggregates of early endocytic protein Ede1-GFP. This is the first identification of a UBX domain-containing protein in clathrin-mediated endocytosis. Tda2 appears at the tail end of each endocytic site, suggesting a function in late stage endocytosis. A Tda2 knockout strain shows similar reductions in bulk and cargo dependent endocytosis through Lucifer yellow and Mup1 uptake assays. Tda2 appears unaffected by clathrin disruption, but is no longer recruited to the endocytic site when cells are treated with the actin depolymerizing agent LatA, suggesting it is associated with the actin cytoskeleton. A crystal structure of Tda2 reveals it is a homolog of mammalian dynein light chain TcTex-1. Tda2 is associated with a larger protein complex in the cytosol but does not co-purify with dynein and is unaffected by addition of the microtubule depolymerizing drug Nocodazole. Tda2 has similar localization to actin capping proteins Cap1/2 which localize to the plus end of actin filaments near the plasma membrane. Tda2 deletion increases Cap1 patch lifetime but reduces its fluorescent intensity. Aim21 fluorescent intensity at endocytic sites is reduced to half without Tda2. When Aim21 is deleted, Tda2 is no longer recruited to endocytic sites and the large Tda2-containing complex is no longer present in the cytosol. Tda2 is a newly identified component of the actin cytoskeleton in stable complex with Aim21. This is the first identification of a TcTex type dynein light chain in yeast and the first dynein light chain associated with clathrin-mediated endocytosis. Thus, we have identified two novel components of endocytic machinery by screening the yeast GFP library. The successful identification of previously uncharacterized endocytic proteins indicates the unique advantages of the GFP library screening. Many previous screens for endocytic proteins rely on the yeast knockout library or cargo accumulation, which have many disadvantages. The GFP library screening method has potential for use with other cellular processes that have distinct cellular localizations and established fluorescent markers. The GFP library also has potential for use in a screen for cargo proteins dependent on clathrin-mediated endocytosis. Additionally, more proteins of the endocytic machinery may be further characterized from the list of Sla1-colocalizing proteins identified in our screen.Item Open Access Regulation of actin capping protein during clathrin-mediated endocytosis(Colorado State University. Libraries, 2022) Lamb, Andrew, author; Di Pietro, Santiago, advisor; Krapf, Diego, committee member; Markus, Steven, committee member; Peersen, Olve, committee memberClathrin-mediated endocytosis (CME) is a major endocytic pathway that is essential in all eukaryotic cells. In the budding yeast S. cerevisiae, polymerization of actin into a branched network is critical to provide the force necessary for membrane invagination during CME. Polymerization of this branched actin network is a highly regulated process, reliant on a multitude of endocytic factors for proper formation. A key regulator is actin capping protein (CP), which binds to the barbed end of actin filaments with high affinity to prevent the loss or addition of actin subunits. While regulation of CP by proteins containing a capping protein-interacting (CPI) motif has been demonstrated in higher eukaryotes, it has not been described in yeast or during endocytosis. Here, we identify and dissect the roles of three CPI motif-containing endocytic factors, Aim21, Bsp1 and Twf1, in CP regulation. Aim21 was the first CPI motif we identified, and the first CPI motif described in yeast. Together with its binding partner Tda2, Aim21 binds to CP through its CPI motif with nanomolar affinity. We demonstrate that Tda2 functions as a dimerization engine for Aim21, bringing two molecules of Aim21 together to form a hetero-tetrameric complex that we term the Tda2-Aim21 complex. Formation of the Tda2-Aim21 complex is essential for a strong interaction with CP, as Aim21 alone binds to CP with more than a 10 fold weaker affinity. Mutating the CPI motif of Aim21 in the yeast genome leads to a recruitment defect in CP and an over-accumulation of F-actin at CME sites, suggesting Aim21 aids in the recruitment of CP to endocytic sites. The little-studied endocytic factor, Bsp1, displays the same phenotype when its CPI motif is mutated in yeast. In addition, the Bsp1 and Aim21 CPI motifs allosterically inhibit the capping function of CP during in vitro actin polymerization assays. When mutations to both the Aim21 and Bsp1 CPI motifs are combined in yeast, CP localization to CME sites is severely reduced, demonstrating that Aim21 and Bsp1 have redundant functions during yeast CME in recruiting a transiently active CP to cortical actin patches. In contrast, the well-conserved actin disassembly factor, twinfilin (Twf1), is not important for recruitment of CP, but is itself reliant on its interaction with CP to localize to CME sites. While the CPI motifs of Aim21 and Bsp1 inhibit the capping function of CP, the Twf1 CPI motif has no effect, despite binding to CP with nanomolar affinity. Mutation of the Twf1 CPI motif results in an accumulation of CP and F-actin at endocytic sites, suggesting that it functions downstream of CP recruitment to recycle CP and actin network components. Together, these findings shed light on how CPI motifs regulate CP in in a step-wise manner during yeast endocytosis.Item Open Access The discovery of novel proteins regulating melanosome biogenesis and function(Colorado State University. Libraries, 2022) Detry, Anna, author; Di Pietro, Santiago, advisor; Hansen, Jeffrey, committee member; Hoerndli, Frederic, committee memberMelanosomes are lysosomal related organelles found in cells which are responsible for making pigment such as skin melanocytes. They are membrane bound organelles that form from the endosomal pathway and have specific proteins and enzymes which allow them to perform the function of melanin production. The process of melanosome biogenesis involves the melanosomes developing through four stages that are classified by electron microscopy appearance. The different melanosome stages have amyloid fibrils formed by proteolytically processed PMEL protein, different amounts of melanin, and different melanosomal proteins. In addition to melanosome biogenesis, another key factor in proper melanin formation and pigmentation is the melanosome luminal pH. The melanin producing enzyme tyrosinase is a pH dependent enzyme. When melanosomes are more acidic, tyrosinase is less functional, leading to less melanin production and a hypopigmentation phenotype. The Di Pietro lab and others have shown that the Two Pore Channel Two (TPC2) is a key regulator of melanosome pH, as well as a regulator of melanosome size and localizes to melanosome membranes. A proximity-dependent biotin identification experiment was preformed using TPC2 and eight potential melanosome proteins were identified. Each of these candidate proteins were knocked down in a human melanoma cell line using small interfering RNA and studied for a potential pigmentation phenotype. Tetraspanin10, phospholipase D1, myosin heavy chain 9, and myosin heavy chain 10 all showed a hypopigmentation phenotype. Two independent tetraspanin 10 knockout cell lines were generated using CRISPR-Cas9 which reproduced the hypopigmentation phenotype. In addition, the phenotype was rescued by re-expressing tetraspanin 10 in the knockout cells and overexpressing tetraspanin 10 in wild type cells showed a hyperpigmentation phenotype. This shows that tetraspanin 10 is involved in the pigmentation process. CD63 is another tetraspanin known to play vital roles in melanosome biogenesis and based on the minimal information aviable on tetraspanin 10, it can be hypothesized as being involved in PMEL processing. The discovery that tetraspanin 10 is involved in skin pigmentation will lead to better understanding of the pigmentation process and pigmentation related diseases.