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

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.

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

Biochemical Journal ◽

10.1042/bcj20160324 ◽

2016 ◽

Vol 473 (17) ◽

pp. 2611-2621 ◽

Cited By ~ 15

Author(s):

Shigeyuki Fujimoto ◽

Tokuji Tsuji ◽

Takashi Fujiwara ◽

Taka-aki Takeda ◽

Chengfeng Merriman ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Secretory Pathway ◽

Pivotal Role ◽

Specific Alkaline Phosphatase ◽

Early Secretory Pathway ◽

Loop 2 ◽

Step Mechanism

Zinc activates tissue-non-specific alkaline phosphatase (TNAP) through a two-step mechanism involving ZnT5–ZnT6 heterodimers and ZnT7 homodimers. We show that the PP-motif in luminal loop 2 of hZnT5, hZnT7 and hZnT5 orthologue CDF5 plays a role in TNAP activation in the early secretory pathway.

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Detailed analyses of the crucial functions of Zn transporter proteins in alkaline phosphatase activation

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012610 ◽

2020 ◽

Vol 295 (17) ◽

pp. 5669-5684 ◽

Cited By ~ 1

Author(s):

Eisuke Suzuki ◽

Namino Ogawa ◽

Taka-aki Takeda ◽

Yukina Nishito ◽

Yu-ki Tanaka ◽

...

Keyword(s):

Secretory Pathway ◽

Molecular Mechanisms ◽

Genetically Engineered ◽

Minor Role ◽

Activation Process ◽

Early Secretory Pathway ◽

Zip Transporters ◽

A Minor ◽

Phosphatase Activation ◽

Dt40 Cells

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

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Evidence for Segregation of Sphingomyelin and Cholesterol during Formation of Copi-Coated Vesicles

The Journal of Cell Biology ◽

10.1083/jcb.151.3.507 ◽

2000 ◽

Vol 151 (3) ◽

pp. 507-518 ◽

Cited By ~ 167

Author(s):

Britta Brügger ◽

Roger Sandhoff ◽

Sabine Wegehingel ◽

Karin Gorgas ◽

Jörg Malsam ◽

...

Keyword(s):

Cholesterol Synthesis ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Lipid Analysis ◽

Coated Vesicles ◽

Early Secretory Pathway ◽

Golgi Membranes ◽

A Cell ◽

Higher Eukaryotes ◽

Specific Lipid

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

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Neuroprogenitor Cells From Patients With TBCK Encephalopathy Suggest Deregulation of Early Secretory Vesicle Transport

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.803302 ◽

2022 ◽

Vol 15 ◽

Author(s):

Danielle de Paula Moreira ◽

Angela May Suzuki ◽

André Luiz Teles e Silva ◽

Elisa Varella-Branco ◽

Maria Cecília Zorél Meneghetti ◽

...

Keyword(s):

Cell Cycle Progression ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Secretory Vesicle ◽

Psychomotor Retardation ◽

Vesicle Transport ◽

Early Secretory Pathway ◽

Pathogenic Variants ◽

Altered Cell ◽

Induced Pluripotent

Biallelic pathogenic variants in TBCK cause encephaloneuropathy, infantile hypotonia with psychomotor retardation, and characteristic facies 3 (IHPRF3). The molecular mechanisms underlying its neuronal phenotype are largely unexplored. In this study, we reported two sisters, who harbored biallelic variants in TBCK and met diagnostic criteria for IHPRF3. We provided evidence that TBCK may play an important role in the early secretory pathway in neuroprogenitor cells (iNPC) differentiated from induced pluripotent stem cells (iPSC). Lack of functional TBCK protein in iNPC is associated with impaired endoplasmic reticulum-to-Golgi vesicle transport and autophagosome biogenesis, as well as altered cell cycle progression and severe impairment in the capacity of migration. Alteration in these processes, which are crucial for neurogenesis, neuronal migration, and cytoarchitecture organization, may represent an important causative mechanism of both neurodevelopmental and neurodegenerative phenotypes observed in IHPRF3. Whether reduced mechanistic target of rapamycin (mTOR) signaling is secondary to impaired TBCK function over other secretory transport regulators still needs further investigation.

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Faculty Opinions recommendation of Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1030067.363460 ◽

2006 ◽

Author(s):

Jef Boeke

Keyword(s):

Secretory Pathway ◽

Early Secretory Pathway

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Faculty Opinions recommendation of Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1030067.361487 ◽

2006 ◽

Author(s):

Deyou Zheng

Keyword(s):

Secretory Pathway ◽

Early Secretory Pathway

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Faculty Opinions recommendation of MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.5374958.5326056 ◽

2010 ◽

Author(s):

Bruno Goud ◽

Kristine Schauer

Keyword(s):

Secretory Pathway ◽

Mapk Signaling ◽

Functional Screening ◽

Early Secretory Pathway

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Membrane traffic related to endosome dynamics and protein secretion in filamentous fungi

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab004 ◽

2021 ◽

Author(s):

Yujiro Higuchi

Keyword(s):

Filamentous Fungi ◽

Protein Secretion ◽

Secretory Pathway ◽

Molecular Mechanisms ◽

Membrane Traffic ◽

Early Endosome ◽

Protein Glycosylation ◽

Hyphal Cell ◽

Cell Factories ◽

Membrane Contacts

ABSTRACT In eukaryotic cells, membrane-surrounded organelles are orchestrally organized spatiotemporally under environmental situations. Among such organelles, vesicular transports and membrane contacts occur to communicate each other, so-called membrane traffic. Filamentous fungal cells are highly polarized and thus membrane traffic is developed to have versatile functions. Early endosome (EE) is an endocytic organelle that dynamically exhibits constant long-range motility through the hyphal cell, which is proven to have physiological roles, such as other organelle distribution and signal transduction. Since filamentous fungal cells are also considered as cell factories, to produce valuable proteins extracellularly, molecular mechanisms of secretory pathway including protein glycosylation have been well investigated. In this review, molecular and physiological aspects of membrane traffic especially related to EE dynamics and protein secretion in filamentous fungi are summarized, and perspectives for application are also described.

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