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

Abstract

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo-to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo-to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.

Sortilin mediates vascular calcification via its recruitment into extracellular vesicles

“…The S825 phosphomimetic activated TNAP activity through TGN localization, while the phosphor-null variant localized to lysosomes. These data agree with previous findings that TNAP activation occurs in the early secretory pathway and subsequently moves and localizes to the plasma membrane (26) and that the sortilin intracellular domain controls the fate of interacting partners from the lysosomes to lipid rafts (47). The p825 site enhances the binding of sortilin-to-Golgi-associated, γ-adaptin ear-containing, ARF-binding protein (GGA) -cytosolic adaptors mediating the sorting of transmembrane proteins (48).…”

“…Trans-Golgi network-to-plasma (TGN-to-plasma) membrane transport requires Rab11, which regulates the transport of recycling endosomes (25). TNAP activates in the Golgi apparatus (26). This consideration engendered the hypothesis that Rab11 regulates sortilin-dependent calcification through trafficking sortilin and activated TNAP from the Golgi to the plasma membrane for Further experiments aimed to determine the relative contribution of Fam20C and CK2 in phosphorylating sortilin on Ser825 in calcifying hSMCs.…”

Biochemical Characterization of Human ZIP13 Protein

“…In addition to homo-oligomerization, the role of the hetero-oligomerization of transporters has been discussed (26,27,37,38). The SLC30A/ZnT (zinc transporter) family members ZnT5 and ZnT6 form a hetero-dimer, and this state is important for the Zn 2ϩ efflux that leads to alkaline phosphatase activation (37,39). The band at the dimer position appeared under non-reducing conditions (Fig.…”

Cellular function and molecular structure of ecto-nucleotidases

“…In a second step, Zn 2+ is loaded onto the protein, converting it from the apo-to the holo-form. The catalytically active holo-form is then trafficked to the plasma membrane [480]. In the absence of ZnT5/ZnT6 heterodimers, the apo-form is degraded [481].…”