The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.
Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Shigeyuki
S
Fujimoto
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Tokuji
T
Tsuji
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Takashi
T
Fujiwara
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Taka-Aki
TA
Takeda
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD 21205, U.S.A.
Chengfeng
C
Merriman
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Ayako
A
Fukunaka
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Yukina
Y
Nishito
Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD 21205, U.S.A.
Dax
D
Fu
Department of Physiology, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel.
Eitan
E
Hoch
Department of Physiology, Faculty of Health Sciences, Ben-Gurion University, Beer-Sheva 84105, Israel.
Israel
I
Sekler
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Kazuhisa
K
Fukue
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Yusaku
Y
Miyamae
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Seiji
S
Masuda
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan.
Masaya
M
Nagao
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan kambe1@kais.kyoto-u.ac.jp.
Taiho
T
Kambe
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002645:Chickens
D002352:Carrier Proteins; C017800:zinc-binding protein
PP-motif; TNAP; ZnT; early secretory pathway; zinc transport; zinc-requiring enzyme