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Kobayashi H.,Laboratory of Membrane Trafficking Mechanisms | Fukuda M.,Laboratory of Membrane Trafficking Mechanisms
Communicative and Integrative Biology | Year: 2013

Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factorstimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes. © 2013 Landes Bioscience. Source


Matsui T.,Laboratory of Membrane Trafficking Mechanisms | Fukuda M.,Laboratory of Membrane Trafficking Mechanisms
EMBO Reports | Year: 2013

Autophagy is an evolutionarily conserved catabolic mechanism that targets intracellular molecules and damaged organelles to lysosomes. Autophagy is achieved by a series of membrane trafficking events, but their regulatory mechanisms are poorly understood. Here, we report small GTPase Rab12 as a new type of autophagic regulator that controls the degradation of an amino-acid transporter. Knockdown of Rab12 results in inhibition of autophagy and in increased activity of mTORC1 (mammalian/mechanistic target of rapamycin complex 1), an upstream regulator of autophagy. We also found that Rab12 promotes constitutive degradation of PAT4 (proton-coupled amino-acid transporter 4), whose accumulation in Rab12-knockdown cells modulates mTORC1 activity and autophagy. Our findings reveal a new mechanism of regulation of mTORC1 signalling and autophagy, that is, quality control of PAT4 by Rab12. © 2013 European Molecular Biology Organizatio. Source


Ohbayashi N.,Laboratory of Membrane Trafficking Mechanisms | Maruta Y.,Laboratory of Membrane Trafficking Mechanisms | Ishida M.,Laboratory of Membrane Trafficking Mechanisms | Fukuda M.,Laboratory of Membrane Trafficking Mechanisms
Journal of Cell Science | Year: 2012

Melanoregulin (Mreg), a product of the dilute suppressor gene, has been implicated in the regulation of melanosome transport in mammalian epidermal melanocytes, given that Mreg deficiency was found to restore peripheral melanosome distribution from perinuclear melanosome aggregation in Rab27A-deficient melanocytes. However, the function of Mreg in melanosome transport has remained unclear. Here, we show that Mreg regulates microtubule-dependent retrograde melanosome transport through the dynein- dynactin motor complex. Mreg interacted with the C-terminal domain of Rab-interacting lysosomal protein (RILP) and formed a complex with RILP and p150Glued (also known as dynactin subunit 1, DCTN1), a component of the dynein-dynactin motor complex, in cultured cells. Overexpression of Mreg, RILP or both, in normal melanocytes induced perinuclear melanosome aggregation, whereas knockdown of Mreg or functional disruption of the dynein-dynactin motor complex restored peripheral melanosome distribution in Rab27A-deficient melanocytes. These findings reveal a new mechanism by which the dynein-dynactin motor complex recognizes Mreg on mature melanosomes through interaction with RILP and is involved in the centripetal movement of melanosomes. © 2012. Source

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