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Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

Hong-Hermesdorf A, Miethke M, Gallaher SD, Kropat J, Dodani SC, Chan J, Barupala D, Domaille DW, Shirasaki DI, Loo JA, Weber PK, Pett-Ridge J, Stemmler TL, Chang CJ, Merchant SS - Nat. Chem. Biol. (2014)

Bottom Line: Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures.Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1.Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA.

ABSTRACT
We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

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Zn deficiency induces CRR1-dependent Cu hyperaccumulationICP-MS analysis of Cu and Zn contents in cells grown under Zn-limited (−Zn) or -replete (+Zn) conditions. (a) Cellular Cu contents in cells from three independent C. reinhardtii cultures (strain CC-4532) that were grown to mid-logarithmic phase in either −Zn or +Zn (containing 2.5 μM ZnCl2) TAP medium (in first (1) and second (2) round of transfer to indicated medium). Quantifications of Cu (b) and Zn (d) contents in cells grown under a concentration range of supplemented Cu (0, 0.02, 0.05, 0.2, 0.5, 2, 5, 10, 25, and 50 μM) in −Zn or +Zn TAP medium. The inset in b is the rescaled representation of Cu contents in the Zn-replete cells. The difference in total Cu contents between −Zn and +Zn cells is about 20-fold. (c) Cu contents in crr1-2 frameshift mutants16 (CC-3960) and CRR1 complemented strains (a and b representing two independent isolates). Quantification results from three different experiments per strain and condition are shown as separate data points (a) or as averages with corresponding standard deviations (b–d).
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Figure 1: Zn deficiency induces CRR1-dependent Cu hyperaccumulationICP-MS analysis of Cu and Zn contents in cells grown under Zn-limited (−Zn) or -replete (+Zn) conditions. (a) Cellular Cu contents in cells from three independent C. reinhardtii cultures (strain CC-4532) that were grown to mid-logarithmic phase in either −Zn or +Zn (containing 2.5 μM ZnCl2) TAP medium (in first (1) and second (2) round of transfer to indicated medium). Quantifications of Cu (b) and Zn (d) contents in cells grown under a concentration range of supplemented Cu (0, 0.02, 0.05, 0.2, 0.5, 2, 5, 10, 25, and 50 μM) in −Zn or +Zn TAP medium. The inset in b is the rescaled representation of Cu contents in the Zn-replete cells. The difference in total Cu contents between −Zn and +Zn cells is about 20-fold. (c) Cu contents in crr1-2 frameshift mutants16 (CC-3960) and CRR1 complemented strains (a and b representing two independent isolates). Quantification results from three different experiments per strain and condition are shown as separate data points (a) or as averages with corresponding standard deviations (b–d).

Mentions: C. reinhardtii keeps intracellular Cu content relatively constant between ~ 1–2.5 × 107 atoms per cell when the external milieu contains chelated Cu ranging from 1 to 80 μM12, but this fine-tuned homeostatic mechanism is disrupted in Zn-limited cells17. Zn-limited cells showed a growth phenotype, especially in the second round of cultivation in limited medium (Supplementary Results, Supplementary Fig. 1). Inductively coupled plasma mass spectrometry (ICP-MS) analysis (Fig. 1) of these cells showed dramatic accumulation of Cu, up to ~ 30×107 atoms per cell in standard growth medium containing 2 μM Cu2+-EDTA (Fig. 1a). If the external supply was increased from 2 to 50 μM, intracellular Cu content was further enhanced to ~ 40 × 107 atoms per cell (Figure 1b). Hyper-accumulation occured only in CRR1 but not crr1 cells, indicating that the pathway is dependent on the nutritional Cu regulon (Fig. 1c). Indeed, the CTR transporters, which are the route for Cu+ assimilation were upregulated in Zn-limited cells despite adequate extracellular Cu2+ and excessive intracellular Cu+ (see below).


Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas.

Hong-Hermesdorf A, Miethke M, Gallaher SD, Kropat J, Dodani SC, Chan J, Barupala D, Domaille DW, Shirasaki DI, Loo JA, Weber PK, Pett-Ridge J, Stemmler TL, Chang CJ, Merchant SS - Nat. Chem. Biol. (2014)

Zn deficiency induces CRR1-dependent Cu hyperaccumulationICP-MS analysis of Cu and Zn contents in cells grown under Zn-limited (−Zn) or -replete (+Zn) conditions. (a) Cellular Cu contents in cells from three independent C. reinhardtii cultures (strain CC-4532) that were grown to mid-logarithmic phase in either −Zn or +Zn (containing 2.5 μM ZnCl2) TAP medium (in first (1) and second (2) round of transfer to indicated medium). Quantifications of Cu (b) and Zn (d) contents in cells grown under a concentration range of supplemented Cu (0, 0.02, 0.05, 0.2, 0.5, 2, 5, 10, 25, and 50 μM) in −Zn or +Zn TAP medium. The inset in b is the rescaled representation of Cu contents in the Zn-replete cells. The difference in total Cu contents between −Zn and +Zn cells is about 20-fold. (c) Cu contents in crr1-2 frameshift mutants16 (CC-3960) and CRR1 complemented strains (a and b representing two independent isolates). Quantification results from three different experiments per strain and condition are shown as separate data points (a) or as averages with corresponding standard deviations (b–d).
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Figure 1: Zn deficiency induces CRR1-dependent Cu hyperaccumulationICP-MS analysis of Cu and Zn contents in cells grown under Zn-limited (−Zn) or -replete (+Zn) conditions. (a) Cellular Cu contents in cells from three independent C. reinhardtii cultures (strain CC-4532) that were grown to mid-logarithmic phase in either −Zn or +Zn (containing 2.5 μM ZnCl2) TAP medium (in first (1) and second (2) round of transfer to indicated medium). Quantifications of Cu (b) and Zn (d) contents in cells grown under a concentration range of supplemented Cu (0, 0.02, 0.05, 0.2, 0.5, 2, 5, 10, 25, and 50 μM) in −Zn or +Zn TAP medium. The inset in b is the rescaled representation of Cu contents in the Zn-replete cells. The difference in total Cu contents between −Zn and +Zn cells is about 20-fold. (c) Cu contents in crr1-2 frameshift mutants16 (CC-3960) and CRR1 complemented strains (a and b representing two independent isolates). Quantification results from three different experiments per strain and condition are shown as separate data points (a) or as averages with corresponding standard deviations (b–d).
Mentions: C. reinhardtii keeps intracellular Cu content relatively constant between ~ 1–2.5 × 107 atoms per cell when the external milieu contains chelated Cu ranging from 1 to 80 μM12, but this fine-tuned homeostatic mechanism is disrupted in Zn-limited cells17. Zn-limited cells showed a growth phenotype, especially in the second round of cultivation in limited medium (Supplementary Results, Supplementary Fig. 1). Inductively coupled plasma mass spectrometry (ICP-MS) analysis (Fig. 1) of these cells showed dramatic accumulation of Cu, up to ~ 30×107 atoms per cell in standard growth medium containing 2 μM Cu2+-EDTA (Fig. 1a). If the external supply was increased from 2 to 50 μM, intracellular Cu content was further enhanced to ~ 40 × 107 atoms per cell (Figure 1b). Hyper-accumulation occured only in CRR1 but not crr1 cells, indicating that the pathway is dependent on the nutritional Cu regulon (Fig. 1c). Indeed, the CTR transporters, which are the route for Cu+ assimilation were upregulated in Zn-limited cells despite adequate extracellular Cu2+ and excessive intracellular Cu+ (see below).

Bottom Line: Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures.Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1.Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California, USA.

ABSTRACT
We identified a Cu-accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulates Cu, a process dependent on the nutritional Cu sensor CRR1, but it is functionally Cu deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. Nano-secondary ion MS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy was consistent with Cu(+) accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu(+) became bioavailable for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mismetallation during Zn deficiency and enabling efficient cuproprotein metallation or remetallation upon Zn resupply.

Show MeSH
Related in: MedlinePlus