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

Abstract

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.

Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

“…Immunofluorescence-Immunostaining for FLAG-hZnT1, hZnT2-HA, hZnT3-Myc, hZnT4-HA, or mZnT8-HA expressed in TKO cells was performed as described previously (20). Briefly, the cells were stained with anti-HA antibody (1:50; Sigma) for hZnT4-HA, with anti-HA tag antibody (1:100; MBL, Nagoya, Japan) for hZnT2-HA and mZnT8-HA, with anti-Myc tag antibody (1:100; MBL) for hZnT3-Myc, or with anti-FLAG tag antibody (anti-DDDDK; 1:100; MBL) for FLAG-hZnT1, followed by Alexa 488-conjugated goat anti-rabbit IgG (1:250; Molecular Probes, Eugene, OR), or with anti-GM130 antibody (1:100; BD Transduction Laboratories), followed by Alexa 594-conjugated goat anti-mouse IgG (1:250; Molecular Probes).…”

“…There is, however, almost no information about the activation step of these enzymes in the secretory process. Using a gene disruption/reexpression system in chicken DT40 cells, and ALPs as marker proteins, we have shown that two ZnT complexes (ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers) activate zinc-requiring enzymes that have matured during the secretory process, converting them from the apo-to the holo-form (7,8,19,20). We used both placenta ALP and tissue nonspecific ALP (TNAP) because their activities are wholly dependent on zinc.…”

Zinc and diabetes

“…ZnT6 mRNA was detected in the islets of Langerhans [34], its functional heterodimerization with ZnT5 has been described [35], and its presence in purified β cells from both mouse [36] and human [37,38] confirmed by massive parallel sequencing (RNASeq).…”

A Dominant Negative Heterozygous G87R Mutation in the Zinc Transporter, ZnT-2 (SLC30A2), Results in Transient Neonatal Zinc Deficiency

“…2D). Moreover, based on the high evolutionary conservation (data not shown) along with the putative homodimer model interface (formed by superimposing the one modeled monomer onto one interacting YiiP monomer), ZnT-2 is likely to form a homodimer that is similar to multiple zinc transporters that have been shown to form homodimeric or heterodimeric structures including ZnT-3, ZnT-5, ZnT-6, and ZnT-8 (42,44,45).…”

“…First, the zinc transporters ZnT-3, ZnT-4, and ZnT-8 which are close homologues of ZnT-2, form homodimers or heterodimers (42,44,45). Second, there is high evolutionary conservation along the putative interface of the homodimeric ZnT-2 model, which was created based on the crystal structure of the E. coli homodimeric zinc transporter YiiP.…”