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Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP.

Ishida M, Ohbayashi N, Fukuda M - Sci Rep (2015)

Bottom Line: Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes.Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes.Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP-kinesin-1 mechanism is responsible for the transport of both.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.

ABSTRACT
Melanosomes are lysosome-related organelles in melanocytes that are transported from the perinucleus to the cell periphery by coordination between bidirectional (anterograde and retrograde) microtubule-dependent transport and unidirectional actin-dependent transport. Although the molecular machineries that mediate retrograde transport and actin-dependent transport have already been identified, little is known about the anterograde transport complex on microtubules in mammalian cells. Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes. Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes. Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP-kinesin-1 mechanism is responsible for the transport of both.

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Formation of the Rab1A–SKIP–kinesin-1 complex in melan-a cells.Endogenous Rab1A molecules were immunoprecipitated from lysates of melan-a cells with anti-Rab1A specific IgG (lane 3) or control IgG (lane 2). Rab1A, SKIP, KLC2, kinesin heavy chain (KHC), and Arl8b were analyzed by immunoblotting with specific antibodies. The amount of IgG heavy chain used for immunoprecipitation was analyzed with an anti-rabbit IgG antibody (bottom panel). It should be noted that SKIP, KLC2, and KHC (Kif5b) were co-purified with Rab1A, but Arl8b was not. Input shows a 1/140 volume of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers are shown on the left.
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f4: Formation of the Rab1A–SKIP–kinesin-1 complex in melan-a cells.Endogenous Rab1A molecules were immunoprecipitated from lysates of melan-a cells with anti-Rab1A specific IgG (lane 3) or control IgG (lane 2). Rab1A, SKIP, KLC2, kinesin heavy chain (KHC), and Arl8b were analyzed by immunoblotting with specific antibodies. The amount of IgG heavy chain used for immunoprecipitation was analyzed with an anti-rabbit IgG antibody (bottom panel). It should be noted that SKIP, KLC2, and KHC (Kif5b) were co-purified with Rab1A, but Arl8b was not. Input shows a 1/140 volume of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers are shown on the left.

Mentions: In the final set of experiments, we attempted to determine whether Rab1A actually regulates anterograde melanosome transport in melanocytes through formation of a Rab1A–SKIP–kinesin-1 complex. To do so, we first performed a coimmunoprecipitation assay with anti-Rab1A specific antibody in melan-a cells. Consistent with the results of the in vitro binding experiments (Fig. 1A)183233, endogenous Rab1A was co-purified with SKIP, KLC2, and Kif5b molecules from lysates of melan-a cells (top three panels in Fig. 4). Intriguingly, however, Arl8b, another SKIP-binding protein18, was not co-purified with Rab1A at all (fifth panel in Fig. 4), indicating that melanocytes contain two different SKIP–kinesin-1 complexes, i.e., the Rab1A–SKIP–kinesin-1 complex on melanosomes and the Arl8–SKIP–kinesin-1 complex on lysosomes. This observation was highly consistent with the results of the binding experiments shown in Supplementary Fig. S2: the Rab1A-binding site and Arl8b-binding site on SKIP almost completely overlapped. In addition, the results of the competition experiments between Rab1A and Arl8b indicated that Rab1A binding to SKIP-RUN (amino acid residues 1–282) was clearly inhibited by the presence of an increasing amount of Arl8b protein (Fig. 5A), i.e., binding of Arl8b and Rab1A to SKIP is mutually exclusive. Therefore, Arl8b and Rab1A are likely to function as distinct, independent cargo receptors for SKIP–kinesin-1.


Rab1A regulates anterograde melanosome transport by recruiting kinesin-1 to melanosomes through interaction with SKIP.

Ishida M, Ohbayashi N, Fukuda M - Sci Rep (2015)

Formation of the Rab1A–SKIP–kinesin-1 complex in melan-a cells.Endogenous Rab1A molecules were immunoprecipitated from lysates of melan-a cells with anti-Rab1A specific IgG (lane 3) or control IgG (lane 2). Rab1A, SKIP, KLC2, kinesin heavy chain (KHC), and Arl8b were analyzed by immunoblotting with specific antibodies. The amount of IgG heavy chain used for immunoprecipitation was analyzed with an anti-rabbit IgG antibody (bottom panel). It should be noted that SKIP, KLC2, and KHC (Kif5b) were co-purified with Rab1A, but Arl8b was not. Input shows a 1/140 volume of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers are shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4316160&req=5

f4: Formation of the Rab1A–SKIP–kinesin-1 complex in melan-a cells.Endogenous Rab1A molecules were immunoprecipitated from lysates of melan-a cells with anti-Rab1A specific IgG (lane 3) or control IgG (lane 2). Rab1A, SKIP, KLC2, kinesin heavy chain (KHC), and Arl8b were analyzed by immunoblotting with specific antibodies. The amount of IgG heavy chain used for immunoprecipitation was analyzed with an anti-rabbit IgG antibody (bottom panel). It should be noted that SKIP, KLC2, and KHC (Kif5b) were co-purified with Rab1A, but Arl8b was not. Input shows a 1/140 volume of the reaction mixture used for immunoprecipitation. The positions of the molecular mass markers are shown on the left.
Mentions: In the final set of experiments, we attempted to determine whether Rab1A actually regulates anterograde melanosome transport in melanocytes through formation of a Rab1A–SKIP–kinesin-1 complex. To do so, we first performed a coimmunoprecipitation assay with anti-Rab1A specific antibody in melan-a cells. Consistent with the results of the in vitro binding experiments (Fig. 1A)183233, endogenous Rab1A was co-purified with SKIP, KLC2, and Kif5b molecules from lysates of melan-a cells (top three panels in Fig. 4). Intriguingly, however, Arl8b, another SKIP-binding protein18, was not co-purified with Rab1A at all (fifth panel in Fig. 4), indicating that melanocytes contain two different SKIP–kinesin-1 complexes, i.e., the Rab1A–SKIP–kinesin-1 complex on melanosomes and the Arl8–SKIP–kinesin-1 complex on lysosomes. This observation was highly consistent with the results of the binding experiments shown in Supplementary Fig. S2: the Rab1A-binding site and Arl8b-binding site on SKIP almost completely overlapped. In addition, the results of the competition experiments between Rab1A and Arl8b indicated that Rab1A binding to SKIP-RUN (amino acid residues 1–282) was clearly inhibited by the presence of an increasing amount of Arl8b protein (Fig. 5A), i.e., binding of Arl8b and Rab1A to SKIP is mutually exclusive. Therefore, Arl8b and Rab1A are likely to function as distinct, independent cargo receptors for SKIP–kinesin-1.

Bottom Line: Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes.Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes.Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP-kinesin-1 mechanism is responsible for the transport of both.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.

ABSTRACT
Melanosomes are lysosome-related organelles in melanocytes that are transported from the perinucleus to the cell periphery by coordination between bidirectional (anterograde and retrograde) microtubule-dependent transport and unidirectional actin-dependent transport. Although the molecular machineries that mediate retrograde transport and actin-dependent transport have already been identified, little is known about the anterograde transport complex on microtubules in mammalian cells. Here we discovered that small GTPase Rab1A on melanosomes recruits SKIP/PLEKHM2 as a Rab1A-specific effector and that Rab1A, SKIP, and a kinesin-1/(Kif5b+KLC2) motor form a transport complex that mediates anterograde melanosome transport in melanocytes. Interestingly, Arl8, Arf-like small GTPase that also interacts with SKIP, is specifically localized at lysosomes and regulates their anterograde transport in melanocytes. Our findings suggest that the anterograde microtubule-dependent transport of melanosomes and lysosomes are differently regulated by independent cargo receptors, i.e., Rab1A and Arl8, respectively, but that a SKIP-kinesin-1 mechanism is responsible for the transport of both.

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