The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation

Shigeyuki Fujimoto, Tokuji Tsuji, Takashi Fujiwara, Taka Aki Takeda, Chengfeng Merriman, Ayako Fukunaka, Yukina Nishito, Dax Fu, Eitan Hoc, Israel Sekler, Kazuhisa Fukue, Yusaku Miyamae, Seiji Masuda, Masaya Nagao, Taiho Kambe

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2611-2621
Number of pages11
JournalBiochemical Journal
Volume473
Issue number17
DOIs
StatePublished - 2016

Fingerprint

Alkaline Phosphatase
Chemical activation
Tissue
Zinc
Secretory Pathway
Enzymes
Enzyme Activation
Caenorhabditis elegans
Proline
Alanine
Stabilization
Membranes
Defects
Mutation
Proteins

Keywords

  • Early secretory pathway
  • Pp-motif
  • TNAP
  • Zinc transport
  • Zinc-requiring enzyme
  • ZnT

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Fujimoto, S., Tsuji, T., Fujiwara, T., Takeda, T. A., Merriman, C., Fukunaka, A., ... Kambe, T. (2016). The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation. Biochemical Journal, 473(17), 2611-2621. https://doi.org/10.1042/BCJ20160324

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation. / Fujimoto, Shigeyuki; Tsuji, Tokuji; Fujiwara, Takashi; Takeda, Taka Aki; Merriman, Chengfeng; Fukunaka, Ayako; Nishito, Yukina; Fu, Dax; Hoc, Eitan; Sekler, Israel; Fukue, Kazuhisa; Miyamae, Yusaku; Masuda, Seiji; Nagao, Masaya; Kambe, Taiho.

In: Biochemical Journal, Vol. 473, No. 17, 2016, p. 2611-2621.

Research output: Contribution to journalArticle

Fujimoto, S, Tsuji, T, Fujiwara, T, Takeda, TA, Merriman, C, Fukunaka, A, Nishito, Y, Fu, D, Hoc, E, Sekler, I, Fukue, K, Miyamae, Y, Masuda, S, Nagao, M & Kambe, T 2016, 'The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation', Biochemical Journal, vol. 473, no. 17, pp. 2611-2621. https://doi.org/10.1042/BCJ20160324
Fujimoto, Shigeyuki ; Tsuji, Tokuji ; Fujiwara, Takashi ; Takeda, Taka Aki ; Merriman, Chengfeng ; Fukunaka, Ayako ; Nishito, Yukina ; Fu, Dax ; Hoc, Eitan ; Sekler, Israel ; Fukue, Kazuhisa ; Miyamae, Yusaku ; Masuda, Seiji ; Nagao, Masaya ; Kambe, Taiho. / The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation. In: Biochemical Journal. 2016 ; Vol. 473, No. 17. pp. 2611-2621.
@article{f54d32517fe046ec9d4f769d53269e97,
title = "The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation",
abstract = "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.",
keywords = "Early secretory pathway, Pp-motif, TNAP, Zinc transport, Zinc-requiring enzyme, ZnT",
author = "Shigeyuki Fujimoto and Tokuji Tsuji and Takashi Fujiwara and Takeda, {Taka Aki} and Chengfeng Merriman and Ayako Fukunaka and Yukina Nishito and Dax Fu and Eitan Hoc and Israel Sekler and Kazuhisa Fukue and Yusaku Miyamae and Seiji Masuda and Masaya Nagao and Taiho Kambe",
year = "2016",
doi = "10.1042/BCJ20160324",
language = "English (US)",
volume = "473",
pages = "2611--2621",
journal = "Biochemical Journal",
issn = "0264-6021",
publisher = "Portland Press Ltd.",
number = "17",

}

TY - JOUR

T1 - The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation

AU - Fujimoto, Shigeyuki

AU - Tsuji, Tokuji

AU - Fujiwara, Takashi

AU - Takeda, Taka Aki

AU - Merriman, Chengfeng

AU - Fukunaka, Ayako

AU - Nishito, Yukina

AU - Fu, Dax

AU - Hoc, Eitan

AU - Sekler, Israel

AU - Fukue, Kazuhisa

AU - Miyamae, Yusaku

AU - Masuda, Seiji

AU - Nagao, Masaya

AU - Kambe, Taiho

PY - 2016

Y1 - 2016

N2 - 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.

AB - 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.

KW - Early secretory pathway

KW - Pp-motif

KW - TNAP

KW - Zinc transport

KW - Zinc-requiring enzyme

KW - ZnT

UR - http://www.scopus.com/inward/record.url?scp=85009471439&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85009471439&partnerID=8YFLogxK

U2 - 10.1042/BCJ20160324

DO - 10.1042/BCJ20160324

M3 - Article

VL - 473

SP - 2611

EP - 2621

JO - Biochemical Journal

JF - Biochemical Journal

SN - 0264-6021

IS - 17

ER -