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Roles of ARF1 and ARF6 in receptor mediated endocytosis of metal‐binding proteins and cadmium‐metallothionein‐1 (CdMT‐1) toxicity in kidney proximal tubule (PT) cells
Author(s) -
Lee WingKee,
Wolff Natascha A.,
Abouhamed Marouan,
Verroust Pierre J.,
Thevenod Frank
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.1158.12
Subject(s) - endocytosis , endosome , chemistry , endocytic cycle , adp ribosylation factor , microbiology and biotechnology , internalization , receptor mediated endocytosis , intracellular , transferrin , transferrin receptor , receptor , endoplasmic reticulum , biochemistry , golgi apparatus , biology
Filtered metal‐binding proteins, such as transferrin (Tf) or metallothionein‐1 (MT‐1), are apically endocytosed partly via megalin/cubulin in kidney PT where CdMT‐1 internalization causes apoptosis. Small GTPase ARF (ADP‐ribosylation factor) proteins regulate vesicular trafficking. We investigated roles of ARF1 and ARF6, which have been involved in secretory and endocytic pathways, respectively, following Tf, MT‐1 and CdMT‐1 uptake by PT cells. WKPT‐0293 Cl.2 cells derived from rat PT S1‐segment were transfected with hemagglutinin‐tagged wildtype (ARF‐WT) or dominant‐negative (ARF‐DN; ARF1‐T31N, ARF6‐T27N) forms of ARFs. Endogenous ARF1 co‐localized with Rab7 (late endo‐/lysosomal) whereas ARF6 with Rab5a and Rab11 (early and recycling endosomes). Live immunofluorescence staining of megalin showed less surface expression in ARF6‐T27N cells, but not in ARF1‐T31N. Fluorescent Tf mainly accumulated perinuclearly in late endo‐/lysosomes in ARF‐WT but was decreased and/or remained near the plasma membrane in ARF‐DN cells. Intracellular MT‐1 was also reduced in ARF‐DN cells. Moreover, CdMT‐1 toxicity was significantly attenuated in ARF1‐T31N (by 66.1 ± 16.6%, n = 10) or ARF6‐T27N (by 51.0 ± 12.0%, n = 9) cells, compared to respective ARF‐WT. The data support roles for ARF1 and ARF6 in endocytosis and trafficking of Tf/MT‐1 to endo‐/lysosomes and CdMT‐1 toxicity of PT cells. Funding: DFG TH 345/8‐1