Human RME-8 Is Involved in Membrane Trafficking through Early Endosomes

Abstract

ABSTRACT. RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.

DNAJC13 mutations in Parkinson disease

“…To investigate the physiological consequence of the DNAJC13 p.Asn855Ser mutation, we assessed the uptake of transferrin (TF), a membrane receptor ligand that is constitutively internalized and recycled through the endosome pathway (12,13). COS7 cells expressing a GFP control, wild-type (WT) or mutant DNAJC13 were exposed to 568-conjugated TF for 1 h at 48C and either fixed immediately (T0) or after a 40 min incubation at 378C (T40).…”

“…4). In cells overexpressing DNAJC13 mutant, there were also fewer clusters (0.59-fold) compared with GFP control (Fisher's P ¼ 0.04), but those remaining were significantly brighter, that is the clusters covered a larger area (1.71-fold; ANOVA P ¼ 0.02, F 2,6 ¼ 8.64; Fisher's P ¼ 0.01) and had enhanced density (1.94-fold; ANOVA P ¼ 0.02, F 2,6 ¼ 8.19; Fisher's P ¼ 0.01) compared with GFP or GFP-DNAJC13-WT, as a result of increased size or density, indicative of increased endocytosis or more likely decreased trafficking out of the TF-labeled endosomes (12). The exact mechanism of mutant-induced alterations to endosomal trafficking will be the focus of future studies; however, it appears that the p.Asn855Ser mutation in DNAJC13 alters its cellular activity and the regulation of endosomal membrane trafficking.…”

“…The retromer generates clathrin-independent carriers that mediate retrograde targeting of selective cargo from endosomes to the trans-Golgi network (21). DNAJC13 is thus thought to function by coordinating retromer with endosomal clathrin coats, either by regulating clathrin dynamics to allow for the sorting of cargo from clathrin subdomains into the retrograde vesicles or by creating clathrin-free patches on endosomal membranes allowing for sites of retromer-mediated vesicle formation (12,20). Preliminary functional analysis indicates that the expression of DNAJC13 p.Asn855Ser in COS7 cells leads to accumulation of TF in endosomal compartments, suggesting interference with endosomal compartmentalization or trafficking as the pathological mechanism of disease.…”

“…A cDNA clone encoding a GFP-fused human DNAJC13 was a gift from Dr Sekiguchi (Osaka University, Japan) (12). The p.Asn855Ser mutation was inserted by site-directed mutagenesis (primers available on request).…”

Disrupted iron homeostasis causes dopaminergic neurodegeneration in mice

“…However, severe loss-of-function mutations are likely to be lethal before birth (3) and have not been described in humans. Interestingly, SNCA and DNAJC13, mutated in patients with PD, modulate TFR1 trafficking (72)(73)(74). VPS35, another PD disease gene, helps sort TFR1 to recycling endosomes, and insufficiency of its retromer complex causes cellular iron deficiency (75).…”