Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway
Metallothionein and ZnT4 in the Cytoplasm Are Required
for Full Activation of TNAP in the Early Secretory Pathway. PLoS ONE 8(10): e77445. doi:10.1371/journal.pone.0077445
Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway
Shigeyuki Fujimoto
Naoya Itsumura
Tokuji Tsuji
Yasumi Anan
Natsuko Tsuji
Yasumitsu Ogra
Tomoki Kimura
Yusaku Miyamae
Seiji Masuda
Masaya Nagao
Taiho Kambe
Stefano L Sensi, University G. D'Annunzio, Italy
The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membranebound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and reexpression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT12/2MT2/2ZnT42/2 cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT12/2MT2/2ZnT42/2 cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT12/2MT2/2 ZnT42/2 cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.
-
. These authors contributed equally to this work.
Of all transition metals, zinc is the most widely used catalytic
and structural factor in proteins [1,2]. Zinc proteomics predicts
that approximately 10% of proteins encoded in the human
genome have a motif that potentially binds to zinc [3,4]. Among
these proteins, approximately 1000 are enzymes, which are
involved in diverse physiological functions and can be classified
into six major classes [5]. Most zinc enzymes use zinc as a catalytic
component [4], and therefore zinc coordination (metalation)
following zinc transport, trafficking and transfer is fundamental
for enzyme activity. The molecular mechanism for this activation
process, however, remains unclear. Metallothionein (MT) has
been suggested to control the activation of cytoplasmic zinc
enzymes [6] and to play a zinc chaperoning role in in vitro studies
[2,7]. All zinc transport proteins, including zinc transporters
(ZnTs) and ZRT/IRT-related proteins (ZIPs), would potentially
be involved in enzyme activation via zinc transport across the cell
membrane [8,9]. However, at present there is little direct
evidence.
Secretory and membrane-bound zinc enzymes, such as matrix
metalloproteinases, angiotensin-converting enzymes [10], A
disintegrin and metalloproteinase (ADAM) family proteins [11], and
alkaline phosphatase [12], are thought to become functional by
incorporating zinc in the early secretory pathway (ESP) before
reaching their final destination. Thus, zinc transport into the
lumen of the ESP is one of the crucial steps for enzyme activation
[9]. Compared with the well-known activation process of secretory
cuproenzymes by Atox1-ATP7A/ATP7B pathways [1315],
understanding of the activation process of secretory and
membrane-bound zinc enzymes has been less clear. We have
previously shown that the ZnT5-ZnT6 hetero-complex and ZnT7
homo-complex (zinc transport complexes) are employed as zinc
entry routes into the ESP [16,17]. We have also shown that the
zinc transport complexes are indispensable for the activation of
secretory and membrane-bound zinc enzymes by converting them
from the apo to the holo form using tissue non-specific alkaline
phosphatase (TNAP) as a reporter enzyme [18]. However, how
other proteins involved in cellular zinc metabolism affect this
activation process remains unknown [19].
Here, we examined the TNAP activation process by establishing
a series of cells deficient in genes encoding molecules known to be
involved in cytoplasmic zinc metabolism. Specifically, we
disrupted the ZnT1, MT and ZnT4 genes in the cells, whose products play
pivotal roles in the maintenance of cellular zinc homeostasis
[8,15,20] via regulatory mechanisms called zinc buffering and
muffling [21,22]. Using (...truncated)