A di-leucine sorting signal in ZIP1 (SLC39A1) mediates endocytosis of the protein.
It has been demonstrated that the plasma membrane expression of ZIP1 is regulated by endocytic mechanisms. In the zinc-replete condition, the level of surface expressed ZIP1 is low due to the rapid internalization of ZIP1. The present study aimed to identify a sorting signal(s) in ZIP1 that mediated endocytosis of ZIP1. Four potential sorting signals (three di-leucine-and one tyrosine-based) were found by searching the eukaryotic linear motif resource for functional sites in proteins (http://elm.eu.org). Site-directed mutagenesis and immunofluorescence microscopic analyses demonstrated that the di-leucine sorting signal, ETRALL144-149, located in the variable loop region of ZIP1, was required for the ZIP1 internalization and lysosomal degradation. Substitutions of alanines for the di-leucine residues (LL148,149/AA) severely impaired the internalization of ZIP1 and subsequent protein degradation, leading to an accumulation of the mutant ZIP1 on the cell surface, as well as inside the cell. Using chimeric proteins composed of an alpha-chain of interleukin-2 receptor fused to the peptides derived from the variable loop region of ZIP1, we found that the di-leucine sorting signal of ZIP1 was required and sufficient for endocytosis of the chimeric proteins.
Huang
Liping
L
United States Department of Agriculture, Agriculture Research Service, Western Human Nutrition Research Center, Davis, CA, USA. lhuang@whnrc.usda.gov
Kirschke
Catherine P
CP
D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D004705:Endocytosis; D015870:Gene Expression; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D021382:Protein Sorting Signals; D021381:Protein Transport; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D027682:Cation Transport Proteins; D021382:Protein Sorting Signals; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; C403517:SLC39A1 protein, human; D007930:Leucine