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Submitted to: FEBS Journal
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 5/31/2007 Publication Date: 6/11/2007 Citation: Huang, L., Kirschke, C.P. A di-leucine sorting signal in zip1 (slc39a1) mediates endocytosis of the protein. FEDERATION OF EUROPEAN BIOCHEMICAL SOCIETY, 274: 3986-3997, 2007. Interpretive Summary: Zinc can not be translocated across the cell membrane by passive diffusion. Therefore, specialized mechanisms are required for transporting zinc across the plasma membrane and distributing zinc around the cell. Most of members of the ZIP family of zinc transporters function to take up zinc into the cell. It has been demonstrated that the amount of cell membrane expressed ZIP1 is regulated by endocytic mechanisms. In zinc-adequate conditions, the level of surface expressed ZIP1 was low due to the rapid internalization of ZIP1 from the cell membrane. The aim of the present work was to identify signal(s) in the ZIP1 protein that mediated the internalization of ZIP1. Four potential signals including three di-leucine-based and one tyrosine-based sorting signals were found in ZIP1 from a search for functional motifs using the ELM server (http://elm.eu.org). Site-directed mutational analyses and immunofluorescence microscopic assays demonstrated that a di-leucine-based sorting signal, ETRALL144-149, located in the variable loop region between transmembrane domain III and IV of ZIP1 was important for the ZIP1 internalization. Substitutions of alanines for the di-leucine residues (LL148, 149/AA) severely impaired the internalization of ZIP1 from the cell surface and subsequent protein degradation leading to accumulation of the mutant ZIP1 on the cell surface as well as inside the cell. Our results also suggest that this di-leucine sorting signal is required for zinc-induced internalization of ZIP1. In conclusion, the regulatory mechanism of constitutive and zinc-induced internalization and subsequent degradation of ZIP1 is falling into the classical adaptor complex (AP)-mediated endocytic pathway. Technical Abstract: It has been demonstrated that the plasma membrane expression of ZIP1 was regulated by endocytic mechanisms. In zinc-replete conditions, the level of surface expressed ZIP1 was low due to the rapid internalization of ZIP1 from the cytoplasmic membrane. The aim of the present work was to identify sorting signal(s) in the ZIP1 protein that mediated the endocytosis of ZIP1. Four potential sorting signals including three di-leucine-based and one tyrosine-based sorting signals were found in ZIP1 from a search for functional motifs using the ELM server (http://elm.eu.org). Site-directed mutagenesis and immunofluorescence microscopic analyses demonstrated that a di-leucine-based sorting signal, ETRALL144-149, located in the variable loop region between transmembrane domain III and IV of ZIP1 was important for the ZIP1 internalization. Substitutions of alanines for the di-leucine residues (LL148, 149/AA) severely impaired the internalization of ZIP1 from the cell surface and subsequent protein degradation leading to accumulation of the mutant ZIP1 on the cell surface as well as inside the cell. Our results also suggest that this di-leucine sorting signal is required for zinc-induced endocytosis of ZIP1. In conclusion, the regulatory mechanism of constitutive and zinc-induced internalization and subsequent degradation of ZIP1 is falling into the classical adaptor complex (AP)-mediated endocytic pathway. |
