Mechanism and Regulation of Cellular Zinc Transport

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126

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn 2؉ and Cd 2؉ , whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.Zinc is an essential element for all living organisms (1). Zinc chemistry is widely exploited to drive enzymatic catalysis, organize protein structures, and mediate macromolecular interactions (2). In known proteomes, zinc-containing metalloproteins account for ϳ10% of the total proteins (3). Zinc metabolism is also very high. In human, ϳ1% of the total body zinc content is replenished daily by the diet (4). The abundant zinc utilization and its rapid turnover necessitate highly efficient zinc uptake mechanisms by which cells accumulate zinc to a total concentration in the submillimolar range (5). The vast majority of cellular zinc is in complex with specific protein partners (6). Free zinc ions, on the other hand, must be strictly limited in the cytoplasm to prevent cytotoxic side effects (7). Zinc efflux transporters and intracellular buffering systems are evolved to maintain an extremely low level of cytoplasmic free zinc, probably in a femtomolar to picomolar range (8, 9). When a zinc supply is available in the extracellular medium, the free zinc concentrations in the cytoplasm are expected to be many orders of magnitude lower than the extracellular zinc concentrations (10, 11). The inward zinc concentration gradients would provide a powerful chemical driving force to draw extracellular zinc ions into the cytoplasm if a transmembrane zinc conduit would connect the external zinc availability and the high intracellular zinc binding capacity. Such a zinc-specific uptake channel has not heretofore been identified.A common assumption is that cellular zinc uptake is an active process mediated by metal transporters. In mammals, Zrt-, Irt-like protein (ZIP) 2 is the only zinc-specific uptake protein identified thus far (12). Although ZIPs supply zinc to meet cellular needs for growth, aberrant ZIP expressions have been linked to uncontrolled cell growth such as that occurring in cancer (13). Functionally, mammalian ZIPs promote zinc influx into the cytoplasm either from the extracellular medium or from zin...

Section: Discussionmentioning

confidence: 99%

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein, ZIPB*

Lin

Chai

Love

et al. 2010

105

126

“…Zinc homeostasis is therefore dynamically maintained by a variety of transporters and other proteins distributed in distinct cellular compartments. Zinc transport is mediated by two major protein families: the Zip family, which mediates Zn 2ϩ influx, and the ZnTs, 3 which are primarily linked to Zn 2ϩ sequestration into intracellular compartments and are, thereby, involved in lowering cytoplasmic Zn 2ϩ concentrations (2). The ZnT family of proteins consists of 10 known members that play versatile roles in many physiological and pathophysiological aspects of zinc homeostasis (3).…”

Section: Znmentioning

confidence: 99%

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

Ohana¹,

Hoch²,

Keasar³

et al. 2009

Self Cite

169

127

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2؉ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn 2؉ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on

“…Zinc transporter (SLC30A) proteins, also known as CDF/ ZnTs, 2 are responsible for the efflux of cytosolic zinc from cells and the mobilization of zinc into intracellular organelles to remove the excess zinc and to ensure a supply of zinc to zincbinding proteins (1)(2)(3)(4)(5)(6)(7)(8). Recently, more data about the biochemical properties of these transporter molecules have been published, including their substrate specificity, structural conformation, metal transport mechanisms, and cellular or physiological functions (1, 5, 8 -13).…”

mentioning

confidence: 99%

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

Fukunaka

Suzuki

Kurokawa

et al. 2009

102

104

The majority of CDF/ZnT zinc transporters form homooligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zincrequiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.