Melanoregulin regulates retrograde melanosome transport through interaction with the RILP·p150Glued complex in melanocytes

Abstract

SummaryMelanoregulin (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 dyneindynactin 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.

Small GTPase Rab2B and Its Specific Binding Protein Golgi-associated Rab2B Interactor-like 4 (GARI-L4) Regulate Golgi Morphology

“…cDNAs of mouse and human GARI-L4 were similarly amplified by PCR and directly inserted into the pGEM-T Easy vector. After verification of their sequences, human and mouse GARI-L4 cDNAs (gene IDs 149647 and 619288, respectively) were subcloned into the pEGFP-C1 vector, pmStr-C1 vector (30), and/or pEF-T7 tag expression vector (29). The sequences of the oligonucleotides for GARI-L4 are also available from the authors on request.…”

The Rab Interacting Lysosomal Protein (RILP) Homology Domain Functions as a Novel Effector Domain for Small GTPase Rab36

“…Anti-Rab7 rabbit polyclonal antibody and anti-Rab11 mouse monoclonal antibody were from Cell Signaling Technology (Danvers, MA) and BD Biosciences, respectively. Anti-melanoregulin rabbit polyclonal antibody and anti-RILP rabbit polyclonal antibody were prepared as described previously (10). Anti-Rab36 rabbit polyclonal antibody was produced by using purified glutathione S-transferase (GST)-mouse Rab36 (11) and affinity-purified as described previously (12).…”

“…The sequences of the mutagenic oligonucleotides used are also available from the authors on request. The mutant Rab36 fragments were subcloned into the pEF-FLAG tag vector (13) and pEGFP-C1 vector (Clontech-Takara Bio Inc.), and the mutant RILP fragments were subcloned into the pEF-T7 tag vector and pmStr-C1 vector (10).…”

Mon1-Ccz1 activates Rab7 only on late endosome and dissociates from lysosome in mammalian cells

“…The cDNA for CI-MPR (CI-MPR is also known as IGF2R) (Waguri et al, 2006) was used to prepare pCIpremCherry-CIMPR-full. The cDNA for mouse Vps41 was amplified by PCR and subcloned into pmStr-C1 (Ohbayashi et al, 2012). The piggyBac transposon-based expression vector (Yusa et al, 2009) was provided by Allan Bradley (Wellcome Trust Sanger Institute, UK).…”