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Mitsunori Fukuda - One of the best experts on this subject based on the ideXlab platform.

  • The GTPase-deficient Rab27A(Q78L) mutant inhibits melanosome transport in melanocytes through trapping of Rab27A effector protein Slac2-a/Melanophilin in their cytosol: development of a novel melanosome-targetinG tag.
    The Journal of biological chemistry, 2014
    Co-Authors: Morié Ishida, Saki P. Arai, Norihiko Ohbayashi, Mitsunori Fukuda
    Abstract:

    The small GTPase Rab27A is a crucial regulator of actin-based melanosome transport in melanocytes, and functionally defective Rab27A causes human Griscelli syndrome type 2, which is characterized by silvery hair. A GTPase-deficient, constitutively active Rab27A(Q78L) mutant has been shown to act as an inhibitor of melanosome transport and to induce perinuclear aggregation of melanosomes, but the molecular mechanism by which Rab27A(Q78L) inhibits melanosome transport remained to be determined. In this study, we attempted to identify the primary cause of the perinuclear melanosome aggregation induced by Rab27A(Q78L). The results showed that Rab27A(Q78L) is unable to localize on mature melanosomes and that its inhibitory activity on melanosome transport is completely dependent on its binding to the Rab27A effector Slac2-a/Melanophilin. When we forcibly expressed Rab27A(Q78L) on mature melanosomes by using a novel melanosome-targeting tag that we developed in this study and named the MST tag, the MST-Rab27A(Q78L) fusion protein behaved in the same manner as wild-type Rab27A. It localized on mature melanosomes without inducing melanosome aggregation and restored normal peripheral melanosome distribution in Rab27A-deficient cells. These findings indicate that the GTPase activity of Rab27A is required for its melanosome localization but is not required for melanosome transport.

  • Functional characterization of two RAB27A missense mutations found in Griscelli syndrome type 2.
    Pigment cell & melanoma research, 2010
    Co-Authors: Norihiko Ohbayashi, Mitsunori Fukuda, Setareh Mamishi, Koutaro Ishibashi, Yuto Maruta, Babak Pourakbari, Banafshe Tamizifar, Masoud Mohammadpour, Nima Parvaneh
    Abstract:

    Human Griscelli syndrome type 2 (GS-2) is characterized by partial albinism and a severe immunologic disorder as a result of RAB27A mutations. In melanocytes, Rab27A forms a tripartite complex with a specific effector Slac2-a/Melanophilin and myosin Va, and the complex regulates melanosome transport. Here, we report a novel homozygous missense mutation of Rab27A, i.e. K22R, in a Persian GS-2 patient and the results of analysis of the impact of the K22R mutation and the previously reported I44T mutation on protein function. Both mutations completely abolish Slac2-a/Melanophilin binding activity but they affect the biochemical properties of Rab27A differently. The Rab27A(K22R) mutant lacks the GTP binding ability and exhibits cytosolic localization in melanocytes. By contrast, neither intrinsic GTPase activity nor melanosomal localization of Rab27A is affected by the I44T mutation, but the Rab27A(I44T) mutant is unable to recruit Slac2-a/Melanophilin. Interestingly, the two mutations differently affect binding to other Rab27A effectors, Slp2-a, Slp4-a/granuphilin-a, and Munc13-4. The Rab27A(K22R) mutant normally binds Munc13-4, but not Slp2-a or Slp4-a, whereas the Rab27A(I44T) mutant shows reduced binding activity to Slp2-a and Munc13-4 but normally binds Slp4-a.

  • Structural basis for the exclusive specificity of Slac2-a/Melanophilin for the Rab27 GTPases.
    Structure (London England : 1993), 2008
    Co-Authors: Mutsuko Kukimoto-niino, Mitsunori Fukuda, Ayako Sakamoto, Eiko Kanno, Kyoko Hanawa-suetsugu, Takaho Terada, Mikako Shirouzu, Shigeyuki Yokoyama
    Abstract:

    Rab27A is required for actin-based melanosome transport in mammalian skin melanocytes through its interaction with a specific effector, Slac2-a/Melanophilin. Mutations that disrupt the Rab27A/Slac2-a interaction cause human Griscelli syndrome. The other Rab27 isoform, Rab27B, also binds all of the known effectors of Rab27A. In this study, we determined the crystal structure of the constitutively active form of Rab27B complexed with GTP and the effector domain of Slac2-a. The Rab27B/Slac2-a complex exhibits several intermolecular hydrogen bonds that were not observed in the previously reported Rab3A/rabphilin complex. A Rab27A mutation that disrupts one of the specific hydrogen bonds with Slac2-a resulted in the dramatic reduction of Slac2-a binding activity. Furthermore, we generated a Rab3A mutant that acquires Slac2-a binding ability by transplanting four Rab27-specific residues into Rab3A. These findings provide the structural basis for the exclusive association of Slac2-a with the Rab27 subfamily, whereas rabphilin binds several subfamilies, including Rab3 and Rab27.

  • Distinct Rab27A binding affinities of Slp2-a and Slac2-a/Melanophilin: Hierarchy of Rab27A effectors.
    Biochemical and biophysical research communications, 2006
    Co-Authors: Mitsunori Fukuda
    Abstract:

    Abstract The small GTPase Rab27A has recently been shown to regulate melanosome transport in mammalian skin melanocytes through sequentially interacting with two Rab27A effectors, Slac2-a/Melanophilin and Slp2-a. Although Slac2-a and Slp2-a contain a similar N-terminal Rab27A-binding domain (named SHD, Slp homology domain), nothing is known about the functional differences between the Slac2-a SHD and Slp2-a SHD. In this study, the Rab27A-binding affinity of ten putative Rab27A effector proteins has been investigated. It has been found that they could be classified into a low-affinity group (e.g., Slac2-a) and a high-affinity group (e.g., Slp2-a and Slp4-a) based on their Rab27A-binding affinity. Kinetic analysis of the GTP-Rab27A-binding to the SHD of Slp2-a, Slp4-a, and Slac2-a by surface plasmon resonance further indicated that the kinetic parameters of Rab27A for the Slp2-a SHD, Slp4-a SHD, and Slac2-a SHD consisted of a fast association rate constant (3.35 × 104, 2.06 × 104, and 2.11 × 104 M−1 s−1, respectively) and a slow dissociation rate constant (4.48 × 10−4, 3.96 × 10−4, and 2.37 × 10−3 s−1 respectively), and their equilibrium dissociation constants were determined to be 13.4, 19.2, and 112 nM, respectively. Our data suggest that distinct Rab27A binding activities of Slac2-a and Slp2-a ensure the order (or hierarchy) of Rab27A effectors that sequentially function in melanosome transport in melanocytes.

  • Functional analysis of slac2-a/Melanophilin as a linker protein between Rab27A and myosin Va in melanosome transport.
    Methods in enzymology, 2005
    Co-Authors: Taruho S. Kuroda, Takashi Itoh, Mitsunori Fukuda
    Abstract:

    Slac2-a/Melanophilin regulates melanosome transport in mammalian skin melanocytes by linking melanosome-bound Rab27A and an actin-based motor protein, myosin Va. Slac2-a consists of an N-terminal Slp homology domain (SHD), which has been identified as a specific GTP-Rab27-binding domain, a myosin Va-binding domain (MBD) in the middle region, and an actin-binding domain (ABD) at the C-terminus. Mutations in the slac2-a/mlph gene cause the abnormal pigmentation (i.e., perinuclear melanosome aggregation in melanocytes) in human Griscelli syndrome type III and in leaden mice because of the inability to form the tripartite protein complex consisting of Rab27A, Slac2-a, and myosin Va. In this chapter we describe the methods, including in vivo melanosome distribution assay combined with dominant-negative approaches and RNA interference technology, that have been used to analyze the function of Slac2-a in melanosome transport in melanocytes.

Jae Sung Hwang - One of the best experts on this subject based on the ideXlab platform.

  • 16-Kauren-2-beta-18,19-triol inhibits melanosome transport in melanocytes by down-regulation of Melanophilin expression.
    Journal of dermatological science, 2019
    Co-Authors: Cheol Hwan Myung, Jongil Park, Ji Eun Lee, Kyuri Kim, Jeong Ah Lee, Sung Chan Hong, Kyung-min Lim, Jae Sung Hwang
    Abstract:

    Abstract Background Rab27a, Mlph, and MyoVa form a tripartite complex and relate to melanosome distribution. Melanophilin (Mlph) acts as a linker protein between Rab27a and MyoVa. The biological activity and function of 16-kauren on the expression of Mlph has not yet been studied. Objective We examined the effect of 16-kauren on melanosome transport and skin pigmentation. Methods Murine Melan-a melanocytes and SP-1 keratinocytes were used for in vitro analysis. Western blot analysis, quantitative real-time polymerase chain reaction, luciferase assay and immunohistochemical staining in 3D pigmented human skin model were performed. Results We found that 16-kauren inhibits melanosome transport in Melan-a melanocytes without affecting melanin synthesis. Treatment with 16-kauren reduced Melanophilin (Mlph), a key protein in melanosome transport, in Melan-a melanocytes, at both the protein and mRNA levels while it did not affect the expression of Rab27a and MyoVa, the other two key proteins for melanosome transport. Notably, the expression of melanogenic proteins, including tyrosinase, trp1, trp2, and MITF, was not affected by 16-kauren. However, 16-kauren attenuated melanosome distribution in co-culture of Melan-a melanocytes and SP-1 keratinocytes as well as in Melan-a monolayer culture. In further confirmation of the depigmenting effects of 16-kauren on Melanoderm™, a 3D pigmented human skin model, treatment with 16-kauren for 12 days increased the brightness of the tissue as determined by lightness value and reduced the distribution of melanosomes as shown in histological examination. Conclusion These results demonstrated that 16-kauren is a selective modulator of a melangenic target, Mlph expression, and can be employed as a new depigmenting strategy.

  • The absence of Rab27a accelerates the degradation of Melanophilin
    Experimental dermatology, 2019
    Co-Authors: Jongil Park, Ji Eun Lee, Cheol Hwan Myung, Hye Sung Jang, Jae Sung Hwang
    Abstract:

    Melanophilin (Mlph) forms an interaction with Rab27a and the actin-based motor protein MyosinVa (MyoVa) on mature melanosome membranes and the tripartite complex regulates melanosome transport in melanocytes. In this study, we found that Rab27a siRNA decreased Mlph and Rab27a protein levels, but Mlph mRNA levels were not changed. Other Rab27a siRNA sequences also showed the same results. When Rab27a siRNA was treated with melan-a melanocytes, Rab27a protein was decreased within 6 hours and Mlph protein was decreased within 24 hours. To determine whether the absence of Rab27a promotes Mlph degradation, we inhibited protein degradation by treatment with proteasome (MG132) and lysosomal enzyme (E64D and Pepstatin A) inhibitors in melan-a melanocytes. MG132 inhibited the degradation of Mlph, but E64D and Pepstatin A had no effect on Mlph. The absence of Rab27a enhanced ubiquitination of Mlph and induced proteasomal degradation. From these results, we concluded that Mlph interaction with Rab27a is important for Mlph stability and melanosome transport.

  • inhibitory effect of 2 methyl naphtho 1 2 3 de quinolin 8 one on melanosome transport and skin pigmentation
    Scientific Reports, 2016
    Co-Authors: Jongil Park, Ha Yeon Lee, Ji Eun Lee, Cheol Hwan Myung, Jae Sung Hwang
    Abstract:

    Melanosomes are lysosome-related organelles with specialized capabilities of melanin synthesis and movement mediated by the Rab27a-Melanophilin-MyosinVa protein complex. In this study, we found that 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO) induced melanosome aggregation around the nucleus in melan-a melanocytes and in melan-a melanocytes/SP-1 keratinocyte co-cultures without inducing toxicity or changing the melanin content. Western blot and real-time PCR analyses showed that MNQO decreased expression of the Rab27a, Melanophilin and MyosinVa proteins and mRNAs, respectively, in melan-a melanocytes. In a reconstituted human epidermis model, treatment with 0.001% MNQO reduced skin pigmentation. Also, MNQO reduced skin pigmentation in brown guinea pigs induced by UVB irradiation. These results indicated that regulation of melanosome transport may serve as a good target for new skin depigmenting agents and MNQO itself could be a candidate.

  • Identification of Novel Rab27a/Melanophilin Blockers by Pharmacophore-Based Virtual Screening
    Applied Biochemistry and Biotechnology, 2014
    Co-Authors: Jong Young Joung, Ha Yeon Lee, Jongil Park, Jee-young Lee, Byung Ha Chang, Ky-youb Nam, Jae Sung Hwang
    Abstract:

    Melanocytes are unique cells that produce specific melanin-containing intracellular organelles called melanosomes. Melanosomes are transported from the perinuclear area of melanocytes toward the plasma membrane as they become more melanized in order to increase skin pigmentation. In this vesicular trafficking of melanosomes, Rab27a, Melanophilin, and myosin Va play crucial roles in linking melanosomes to actin-based motors. To identify novel compounds to inhibit binding interface between Rab27a and Melanophilin, a pharmacophore model was built based on a modeled 3D structure of the protein complex that describes the essential binding residues in the intermolecular interaction. A pharmacophore model was employed to screen a chemical library database. Finally, 25 virtual hits were selected for biological evaluations. The biological activities of 11 analogues were evaluated in a second assay. Two compounds were identified as having concentration-dependent inhibitory activity. By analyzing structure–activity relationships of derivatives of BMD-20, two hydroxyl functional groups were found to be critical for blocking the intermolecular binding between Rab27a and Melanophilin.

  • identification of novel rab27a Melanophilin blockers by pharmacophore based virtual screening
    Applied Biochemistry and Biotechnology, 2014
    Co-Authors: Jong Young Joung, Ha Yeon Lee, Jongil Park, Jee-young Lee, Byung Ha Chang, Ky-youb Nam, Jae Sung Hwang
    Abstract:

    Melanocytes are unique cells that produce specific melanin-containing intracellular organelles called melanosomes. Melanosomes are transported from the perinuclear area of melanocytes toward the plasma membrane as they become more melanized in order to increase skin pigmentation. In this vesicular trafficking of melanosomes, Rab27a, Melanophilin, and myosin Va play crucial roles in linking melanosomes to actin-based motors. To identify novel compounds to inhibit binding interface between Rab27a and Melanophilin, a pharmacophore model was built based on a modeled 3D structure of the protein complex that describes the essential binding residues in the intermolecular interaction. A pharmacophore model was employed to screen a chemical library database. Finally, 25 virtual hits were selected for biological evaluations. The biological activities of 11 analogues were evaluated in a second assay. Two compounds were identified as having concentration-dependent inhibitory activity. By analyzing structure-activity relationships of derivatives of BMD-20, two hydroxyl functional groups were found to be critical for blocking the intermolecular binding between Rab27a and Melanophilin.

Miguel C. Seabra - One of the best experts on this subject based on the ideXlab platform.

  • myrip uses distinct domains in the cellular activation of myosin va and myosin viia in melanosome transport
    Pigment Cell & Melanoma Research, 2009
    Co-Authors: Jose S Ramalho, Miguel C. Seabra, Abul K Tarafder, V S Lopes, Alistair N. Hume
    Abstract:

    Myrip is a Rab27a and MyosinVIIa (MyoVIIa) linking protein that may regulate melanosome transport in the retinal pigment epithelium (RPE). Myrip also binds MyosinVa (MyoVa) in vitro however it is unclear whether this interaction is of sufficient affinity to be physiologically relevant. Here, we addressed the questions of whether Myrip interacts with MyoVa in cells and the molecular basis of cellular activation of MyoVa and MyoVIIa by Myrip. To answer these questions we used melanosome transport in skin melanocytes and RPE cells as read-outs of MyoVa and MyoVIIa activity. We found that Myrip recruits and activates MyoVa on skin melanosomes with similar efficiency to the established MyoVa activator Melanophilin (Mlph). Mutagenesis showed that a Myrip-Mlph conserved amphipathic helix (MMAH) is essential for MyoVa interaction while other Myrip regions, including the MyoVa exon F binding domain equivalent, play non-essential roles in this interaction. This suggests that, in contrast to Mlph, Myrip interacts with MyoVa lacking melanocyte-specific exon F. Parallel studies of RPE melanosome transport reveal that Myrip-specific inserts, but not the MMAH, are essential for MyoVIIa activation. We conclude that Myrip is a versatile Rab27a-associated myosin-activating protein that mediates cellular activation of MyoVa and MyoVIIa via non-overlapping domains.

  • Rab3GEP Is the Non-redundant Guanine Nucleotide Exchange Factor for Rab27a in Melanocytes
    The Journal of biological chemistry, 2008
    Co-Authors: Ana C. Figueiredo, Jose S Ramalho, Abul K Tarafder, Christina Wasmeier, Rudi Baron, Miguel C. Seabra
    Abstract:

    Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting Melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

  • a coiled coil domain of Melanophilin is essential for myosin va recruitment and melanosome transport in melanocytes
    Molecular Biology of the Cell, 2006
    Co-Authors: Alistair N. Hume, Jose S Ramalho, Elena V Sviderskaya, Abul K Tarafder, Miguel C. Seabra
    Abstract:

    Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F-binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

  • the role of rab27a in the regulation of melanosome distribution within retinal pigment epithelial cells
    Molecular Biology of the Cell, 2004
    Co-Authors: Clare E Futter, Jose S Ramalho, G B Jaissle, Mathias W Seeliger, Miguel C. Seabra
    Abstract:

    Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, Melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.

  • Rab GTPases and myosin motors in organelle motility.
    Traffic (Copenhagen Denmark), 2004
    Co-Authors: Miguel C. Seabra, Evelyne Coudrier
    Abstract:

    The actin cytoskeleton is essential to ensure the proper location of, and communication between, intracellular organelles. Some actin-based myosin motors have been implicated in this process, particularly members of the class V myosins. We discuss here the emerging role of the Ras-like GTPases of the Rab family as regulators of myosin function in organelle transport. Evidence from yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab GTPases are crucial components of the myosin organelle receptor machinery. Better understood is the case of the melanosome where Rab27a recruits a specific effector called Melanophilin, which in turn binds myosin Va. The presence of a linker protein between a Rab and a myosin may represent a general mechanism. We argue that Rabs are ideally suited to perform this role as they are exquisite organelle markers. Furthermore, the molecular switch property of Rabs may enable them to regulate the timing of the myosin association with the target organelle.

Taruho S. Kuroda - One of the best experts on this subject based on the ideXlab platform.

  • Functional analysis of slac2-a/Melanophilin as a linker protein between Rab27A and myosin Va in melanosome transport.
    Methods in enzymology, 2005
    Co-Authors: Taruho S. Kuroda, Takashi Itoh, Mitsunori Fukuda
    Abstract:

    Slac2-a/Melanophilin regulates melanosome transport in mammalian skin melanocytes by linking melanosome-bound Rab27A and an actin-based motor protein, myosin Va. Slac2-a consists of an N-terminal Slp homology domain (SHD), which has been identified as a specific GTP-Rab27-binding domain, a myosin Va-binding domain (MBD) in the middle region, and an actin-binding domain (ABD) at the C-terminus. Mutations in the slac2-a/mlph gene cause the abnormal pigmentation (i.e., perinuclear melanosome aggregation in melanocytes) in human Griscelli syndrome type III and in leaden mice because of the inability to form the tripartite protein complex consisting of Rab27A, Slac2-a, and myosin Va. In this chapter we describe the methods, including in vivo melanosome distribution assay combined with dominant-negative approaches and RNA interference technology, that have been used to analyze the function of Slac2-a in melanosome transport in melanocytes.

  • identification and biochemical analysis of slac2 c myrip as a rab27a myosin va viia and actin binding protein
    Methods in Enzymology, 2005
    Co-Authors: Taruho S. Kuroda, Mitsunori Fukuda
    Abstract:

    Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/Melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

  • the actin binding domain of slac2 a Melanophilin is required for melanosome distribution in melanocytes
    Molecular and Cellular Biology, 2003
    Co-Authors: Taruho S. Kuroda, Hiroyoshi Ariga, Mitsunori Fukuda
    Abstract:

    Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/Melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (DeltaABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

  • slac2 a Melanophilin the missing link between rab27 and myosin va implications of a tripartite protein complex for melanosome transport
    Journal of Biological Chemistry, 2002
    Co-Authors: Mitsunori Fukuda, Taruho S. Kuroda, Katsuhiko Mikoshiba
    Abstract:

    Abstract Myosin Va is a member of the unconventional class V myosin family, and a mutation in themyosin Va gene causes pigment granule transport defects in human Griscelli syndrome and dilute mice. How myosin Va recognizes its cargo (i.e. melanosomes), however, has re- mained undetermined over the past decade. In this study, we discovered Slac2-a/Melanophilin to be the “missing link” between myosin Va and GTP-Rab27A present in the melanosome. Deletion analysis and site-directed mutagenesis showed that the N-terminal Slp (synaptotagmin-like protein) homology domain of Slac2-a specifically binds Rab27A/B isoforms and that the C-terminal half directly binds the globular tail of myosin Va. The tripartite protein complex (Rab27A· Slac2-a·myosin Va) in melanoma cells was further confirmed by immunoprecipitation. The discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.

Tetsuro Izumi - One of the best experts on this subject based on the ideXlab platform.

  • Melanophilin Accelerates Insulin Granule Fusion without Predocking to the Plasma Membrane.
    Diabetes, 2020
    Co-Authors: Hao Wang, Kouichi Mizuno, Noriko Takahashi, Eri Kobayashi, Jun Shirakawa, Yasuo Terauchi, Haruo Kasai, Katsuhide Okunishi, Tetsuro Izumi
    Abstract:

    Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that Melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both Melanophilin-mutated leaden mouse and Melanophilin-downregulated human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca2+]i rise, Melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.

  • Structure of the small GTPase Rab27b shows an unexpected swapped dimer
    Acta Crystallographica Section D Biological Crystallography, 2007
    Co-Authors: Leonard M. G. Chavas, Seiji Torii, Tetsuro Izumi, Hironari Kamikubo, Masato Kawasaki, Kentaro Ihara, Ryuichi Kato, Mikio Kataoka, Soichi Wakatsuki
    Abstract:

    Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of Rab27a and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as Melanophilin and granuphilin, both Rab27a and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.

  • The roles of Rab27 and its effectors in the regulated secretory pathways.
    Cell structure and function, 2003
    Co-Authors: Tetsuro Izumi, Hiroshi Gomi, Kazuo Kasai, Shin Mizutani, Seiji Torii
    Abstract:

    Regulated secretory pathways are highly developed in multicellular organisms as a means of intercellular communication. Each of these pathways harbors unique store organelles, such as granules in endocrine and exocrine tissues and melanosomes in melanocytes. It has recently been shown that the monomeric GTPase Rab27 subfamily regulates the exocytosis of these cell-specific store organelles. Furthermore, genetic alterations of Rab27a cause Griscelli syndrome in humans that manifests as pigmentary dilution of the skin and the hair and variable immunodeficiency due to defects in the transport of melanosomes in melanocytes and lytic granules in cytotoxic T-lymphocytes. Rab27 acts through organelle-specific effector proteins, such as granuphilin in pancreatic beta cells and Melanophilin in melanocytes. The Rab27 and effector complex then interacts with proteins that are essential for membrane transport and fusion, such as syntaxin 1a and Munc18-1 for granuphilin and myosin Va for Melanophilin. Genome information suggests that other putative Rab27 effector proteins, tentatively termed as exophilins or Slp/Slac2, are predicted to exist because these proteins share the conserved N-terminal Rab27-binding domain and show Rab27-binding activity in vitro or when overexpressed in cell lines. These findings suggest that the Rab27 subfamily regulates various exocytotic pathways using multiple organelle-specific effector proteins.

  • Melanophilin directly links rab27a and myosin va through its distinct coiled coil regions
    FEBS Letters, 2002
    Co-Authors: Kazuaki Nagashima, Seiji Torii, Michihiro Igarashi, Koichi Okamoto, Toshiyuki Takeuchi, Tetsuro Izumi
    Abstract:

    Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melano- philin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat-color mutation genes, dilute (MyoVa d ), ashen (Rab27a ash ), and leaden (Mlph ln ), act in the same or overlapping pathways. Here we have cloned and characterized a human Melanophilin homolog, which belongs to the rabphilin3/granuphilin-like Rab effector family. Cosedimen- tation assays using recombinant proteins reveal that melano- philin directly binds to Rab27a and myosin Va through its N-terminal and its first C-terminal coiled-coil region, respec- tively. Moreover, we show that Rab27a, Melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV-II. These findings suggest that Melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab-binding region conserved in a novel rabphilin3/ granuphilin-like Rab effector family constitutes an K-helix- based coiled-coil structure. fl 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

  • Melanophilin directly links Rab27a and myosin Va through its distinct coiled-coil regions.
    FEBS letters, 2002
    Co-Authors: Kazuaki Nagashima, Seiji Torii, Michihiro Igarashi, Koichi Okamoto, Toshiyuki Takeuchi, Tetsuro Izumi
    Abstract:

    Rab GTPases regulate the membrane transport pathways by recruiting their specific effector proteins. Melanophilin, a putative Rab effector, has recently been identified as a gene that is mutated in leaden mice, in which peripheral localization of melanosomes is impaired in melanocytes. Genetic studies suggest that three coat-color mutation genes, dilute (MyoVa(d)), ashen (Rab27a(ash)), and leaden (Mlph(ln)), act in the same or overlapping pathways. Here we have cloned and characterized a human Melanophilin homolog, which belongs to the rabphilin3/granuphilin-like Rab effector family. Cosedimentation assays using recombinant proteins reveal that Melanophilin directly binds to Rab27a and myosin Va through its N-terminal and its first C-terminal coiled-coil region, respectively. Moreover, we show that Rab27a, Melanophilin, and myosin Va form a ternary complex in the human melanocyte cell line HMV-II. These findings suggest that Melanophilin has a role in bridging Rab27a on melanosomes and myosin Va on actin filaments during melanosome transport. We also propose that the Rab-binding region conserved in a novel rabphilin3/granuphilin-like Rab effector family constitutes an alpha-helix-based coiled-coil structure.