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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 α-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.
Intracellular zinc homeostasis is achieved through coordinated regulations of different zinc transporters involved in influx, efflux, and intracellular compartmental sequestration or release. Two families of zinc transporters (SLC30, ZNT and SLC39, ZIP) have been identified in mammals [1–18]. The ZIP members are essential for an increase of cytoplasmic zinc concentrations by enhancement of zinc uptake or release of the stored zinc from subcellular compartments to the cytoplasm of the cell when zinc is deficient . On the other hand, zinc efflux and intracellular compartmentation are accomplished by the members of the ZNT proteins when zinc is in excess .
The ZIP proteins (SLC39A1-14) are predicted to have eight transmembrane domains with an intracellular cytosolic histidine-rich loop (variable loop region) between transmembrane domains III and IV . They share little conservation both in the sequence and the length of the loop in this region, except for the sequence (HX)n where H is the histidine residue, X is usually aspartic acid, glutamic acid, glycine, lysine, asparagines, arginine, or serine, and n generally is in the range 2–5 . The histidine residues in the loop region of ZIP proteins are thought to bind zinc. However, the exact function of the loop region is not understood.
Regulations of the ZIP protein activities have been found to occur at multiple levels, including transcription [20–23] and intracellular protein trafficking [14,24,25]. Intracellular trafficking of ZIP1, ZIP3, ZIP4, and ZIP5 appears to be a regulated process important for maintaining cellular zinc homeostasis [14,24,25]. In zinc-depleted cells, ZIP proteins seem to be internalized more slowly from the plasma membrane, resulting in an accumulation of the activated ZIP proteins on the cell surface, which leads to an increased zinc influx. On the other hand, in zinc-replete cells, these ZIP proteins are rapidly removed from the plasma membrane to intracellular compartments. The internalization of ZIP proteins from the cell surface lowers the amount of proteins available for zinc uptake on the cell surface, which leads to a decrease in zinc influx [24,26]. Endocytosis, recycling, and/or degradation of ZIP proteins contribute to the rapid modulation of the amount of surface zinc uptake proteins in response to the change in cellular zinc concentrations. By changing the relative rate of zinc uptake protein internalization, cells can adjust the intracellular labile zinc pool level promptly.
Many plasma membrane proteins bear their endocytic signals within the cytosolic domains of proteins. These signals, identified by sequence correlations and mutational analyses, are a short stretch of consensus amino acid residues with key residues for their function. These signals are thought to interact with specific recognition molecules to form transport intermediates that sort membrane proteins into different sites within cells . The best understood endocytic signals are the di-leucine ([DER]XXXL[LVI]) and tyrosine-based sorting signals. The di-leucine signals with consensus sequences [DE]XXXL[LI] predominantly target membrane proteins from the cell surface to the endosomal–lysosomal compartments . Most tyrosine-based signals conform to the consensus sequences YXXØ, where X is any amino acid and Ø is an amino acid with a bulky hydrophobic side chain. The tyrosine-based signal is responsible for endocytosis of membrane proteins and direct sorting of membrane proteins to a variety of intracellular compartments . Both [DE]XXXL[LI] and YXXØ can be recognized by heterotetrameric adaptor protein complexes (AP-1, AP-2, and AP-3) with a distinct affinity of interaction in the formation of clathrin–AP coat complexes [27,29,30]. The YXXØ signal can also be recognized by a fourth AP complex (AP-4) for protein sorting [31,32].
The cellular localization of zinc transporters including ZIP1, ZIP3–5, ZNT4, and ZNT6 are regulated in response to the fluctuations of cellular zinc concentrations [6,14,24,25,33]. However, the sorting signals for the intracellular protein trafficking carrying in these transporter proteins are not clear. The present study aimed to identify a sorting signal(s) in ZIP1 that mediated the internalization of ZIP1. Here, we demonstrate that a stretch of six amino acids with a consensus sequence for a di-leucine signal (EXXXLL144–149) in the variable loop region of ZIP1 plays a critical role in mediating ZIP1 endocytosis and protein degradation. We further demonstrate that this internalization signal of ZIP1 is sufficient for the endocytosis of the IL2R-ZIP1 chimeric proteins.
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It has been demonstrated that the extent of intracellular trafficking of ZIP through exocytosis and endocytosis is cell dependent [10,24,35]. Intracellular zinc deficiency reduces the endocytic arm. This facilitates uptake of zinc and restores normal cellular zinc homeostasis. On the other hand, when cellular zinc levels are elevated, the rate of endocytosis of ZIP1 is increased to reduce zinc influx. Previous studies have demonstrated that ZIP1 is constitutively endocytosed from the cell surface and travels to the intracellular compartments in the zinc-adequate condition . In the present study, four potential protein trafficking signals in ZIP1 were revealed by searching the eukaryotic linear motif resource for functional sites in proteins. We demonstrate that a di-leucine sorting signal, ETRALL144−149, located in the variable loop region of ZIP1, which has the consensus sequences of a leucine doublet and an acidic residue at position −4 relative to the first leucine of the leucine doublet, facilitates the endocytosis of ZIP1 and protein degradation. Targeted mutations of the di-leucine residues in this signal led to in an increase in ZIP1 expression on the cell surface. Meanwhile, the mutations of the di-leucine residues resulted in a higher accumulation of ZIP1 within the cell due to the reduction in the lysosomal degradation of ZIP1. The discovery of a trafficking signal in a highly variable region of ZIP1 suggests that members of the ZIP family may utilize different trafficking signals within the proteins for intracellular organelle targeting. Nevertheless, a similar sequence (ESPELL) is found in the corresponding loop region of ZIP4 among the 14 ZIP proteins, suggesting that ZIP4 may undergo a similar trafficking pathway as ZIP1. Moreover, both di-leucine signals in ZIP1 and ZIP4 have another acidic residue further amino-terminal to the EXXXLL motif that adds to the strength of the signal for adaptor protein targeting .
In cultured CHO cells, the steady-state distribution of ZIP1 favors the Golgi localization (Figs 2 and 3). Disruption of the di-leucine signal (LL148,149) had a detrimental effect on the endocytosis of ZIP1 but not on the intracellular trafficking of ZIP1 from the Golgi location to the cell surface, indicating that the signal for the plasma membrane targeting of ZIP1 is distinguished from the signal for plasma membrane retrieval and protein degradation. The signal(s) mediating the ZIP1 exocytotic arm of trafficking remains to be mapped.
The [DE]XXXL[LI] signals in mammalian proteins mediate rapid internalization and targeting to endosomal–lysosomal compartments. The location of a functional di-leucine signal in the histidine-rich loop region of ZIP1 may bear physiological significance because the histidine residues in this region have been long suspected to be bound to zinc and play a role in zinc transport. Given that the sequence (HX)2 is only eight amino acids downstream of the di-leucine signal (LL148,149) and this di-leucine signal is required for the endocytosis of ZIP1, we hypothesize that the (HX)2 in the variable loop region of ZIP1 may function as a sensor for cellular zinc concentrations. The interaction of the adaptor complex bound to the di-leucine signal with zinc bound histidine residues in (HX)2 may be important for regulating the endocytosis rate of ZIP1 and subsequently targeting it to the lysosomal compartment for degradation under the zinc-replete condition.
It appears that signal-based regulation of metal transporters is a universal regulatory mechanism for early responses for the change in cellular metal concentrations. In yeast, the high affinity zinc uptake protein (ZRT1) was rapidly internalized and degraded through an ubiquitin conjugation signal located in the variable loop region of ZRT1 when cells were exposed to high zinc concentrations [26,44]. In mammalian cells, studies have shown that the cellular localization of zinc uptake proteins, including ZIP1, ZIP3, ZIP4, and ZIP5, are regulated in response to the fluctuation of cytoplasmic zinc concentrations [14,24,33]. However, the signal(s) in these proteins that mediate the plasma membrane targeting and retrieval has not been revealed. Identification of a di-leucine signal within ZIP1 that mediated the protein internalization and degradation in the present study highlights a molecular basis for zinc-induced regulations of zinc transporter expression on the cell surface. A similar motif was previously identified in a copper transporter, ATP7A [45–48]. The di-leucine signal (LL147−148) proximal to the C-terminal tail of ATP7A mediates the recycling ATP7A from the plasma membrane to the trans Golgi network (TGN) in nonpolarized cells in the steady-state condition. However, the same signal in ATP7A is also responsible for targeting ATP7A from the TGN to the basal–lateral membrane of polarized cells to facilitate efflux of copper from the cell in a copper elevated condition.
We have previously reported that the protein expression level of ZIP1 in human prostate epithelial cells were down-regulated by zinc . This zinc-induced down-regulation of ZIP1 expression was not associated with the transcriptional activity of the ZIP1 gene . Di-leucine signal-mediated lysosomal targeting and subsequent protein degradation after internalization of the protein have been observed in plasma membrane proteins, including epidermal growth factor receptor , β-site APP cleaving enzyme , and CD3 gamma [51,52]. Our observations that disruption of a functional di-leucine signal in ZIP1 inhibited the endocytosis of ZIP1, resulting in an accumulation of ZIP1 on the cell surface as well as inside the cell (present study), imply that a significant population of ZIP1 travels through the plasma membrane en route to lysosomes for protein degradation when cellular zinc is elevated.
In summary, we have identified a di-leucine protein trafficking signal in the variable loop region of ZIP1. Substitution of alanines for the leucine doublets in this di-leucine signal inhibited the internalization of ZIP1 in CHO cells. Disruption of this endocytic signal also led to an accumulation of ZIP1 within CHO cells.