Glycerol transporter 1 (Gt1) and zinc-regulated transporter 1 (Zrt1) function in different modes for zinc homeostasis in Komagataella phaffii (Pichia pastoris)

Yang, Yankun; Pan, Yingyue; Liu, Guoqiang; Dong, Guibin; Zhan, Chunjun; Koffas, Mattheos A. G.; Liu, Xiuxia; Li, Ye; Liu, Chunli; Bai, Zhonghu

doi:10.1007/s10529-020-02964-4

Glycerol transporter 1 (Gt1) and zinc-regulated transporter 1 (Zrt1) function in different modes for zinc homeostasis in Komagataella phaffii (Pichia pastoris)

Original Research Paper
Published: 13 July 2020

Volume 42, pages 2413–2423, (2020)
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Yankun Yang ORCID: orcid.org/0000-0003-4815-6832^1,2,4^na1,
Yingyue Pan^1,2^na1,
Guoqiang Liu^1,2,
Guibin Dong^1,2,
Chunjun Zhan^1,2,
Mattheos A. G. Koffas³,
Xiuxia Liu^1,2,
Ye Li^1,2,
Chunli Liu^1,2 &
…
Zhonghu Bai^1,2

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Abstract

Objectives

To identify the zinc transport function of the membrane proteins Gt1 and Zrt1 in Komagataella phaffii (Pichia pastoris) and study their regulatory mode.

Results

Two membrane proteins that might have zinc transport function were found in K. phaffii. GT1 was known to encode a glycerol transporter belonging to the Major Facilitator Superfamily. ZRT1 was predicted to resemble the zinc transporter gene in Saccharomyces cerevisiae. Consistent with the prediction, protein plasma-membrane localizations were confirmed by ultracentrifugation and confocal microscopy. Their zinc binding abilities were identified by ITC in vitro, and the impaired zinc uptake activity caused by their deficiencies was confirmed by zinc fluorescence quantification in vivo. Furthermore, zinc excess could turn the two channels off, while zinc deficiency induced their expressions. Gt1 could only function to maintain zinc homeostasis in glycerol, while the block of Gt1 function might lead to Zrt1 upregulation in glucose.

Conclusions

The zinc transport capabilities of Gt1 and Zrt1 were identified in vivo and in vitro. Their regulatory mode to maintain zinc homeostasis in K. phaffii is a new inspiration.

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Abbreviations

MFS:: Major facilitator superfamily
LZM:: Low-zinc medium
ITC:: Isothermal titration calorimetry
ZIF:: Zinc-induced facilitator
ZIFL:: Zinc-induced-facilitator-like

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Acknowledgements

This article was supported by the National Natural Science Foundation of China (31570034, 21908077), national first-class discipline program of Light Industry Technology and Engineering (LITE2018-24), The fifteenth batch of the “Six Talent Peaks Project in Jiangsu Province” (SWYY-180), the 111 Project (111-2-06), and China Scholarship Council.

Supporting information

Supplementary Table 1—List of strains used in this study.

Supplementary Table 2—List of plasmids used and constructed in this study.

Supplementary Table 3—List of primers used in the plasmid construction and quantitative PCR.

Supplementary Table 4—Representative fitted ITC binding data

Supplementary Figure 1—Zrt1 and Gt1 located to the plasma membrane in K. phaffii. a Western blotting of HA-tagged Gt1 and Zrt1. Samples of cytoplasmic (C) fractions, membrane (M) fractions, and total lysate (T) in detergent were detected. b Fluorescence images of recombinant strains containing pGAPZ B (the left panels), pGAP-RFP-Gt1 (the middle panels), or pGAP-RFP-Zrt1 (the right panels) were visualized using confocal scanning laser microscopy. Scale bar = 5 μm

Supplementary Figure 2—Purification of recombinant Gt1 protein and Zrt1 protein.

Supplementary Figure 3—Representative fitted ITC binding profiles for Gt1 (a) and Zrt1 (b) with Zn²⁺. For Zn²⁺ binding to Gt1 and Zrt1, a single binding site is observed for each protein at a 1:1 stoichiometric ligand: protein ratio.

Author information

Yankun Yang and Yingyue Pan contributed equally to this work.

Authors and Affiliations

Key Laboratory of Carbohydrate Chemistry and Biotechnology, School of Biotechnology, Jiangnan University, Wuxi, 214122, China
Yankun Yang, Yingyue Pan, Guoqiang Liu, Guibin Dong, Chunjun Zhan, Xiuxia Liu, Ye Li, Chunli Liu & Zhonghu Bai
National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China
Yankun Yang, Yingyue Pan, Guoqiang Liu, Guibin Dong, Chunjun Zhan, Xiuxia Liu, Ye Li, Chunli Liu & Zhonghu Bai
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180, USA
Mattheos A. G. Koffas
Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, Wuxi, 214122, China
Yankun Yang

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Yankun Yang
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Yingyue Pan
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Guoqiang Liu
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Guibin Dong
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Mattheos A. G. Koffas
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Xiuxia Liu
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Ye Li
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Chunli Liu
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Zhonghu Bai
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Contributions

YY and YP designed and carried out the most experimental work and drafted the manuscript. GL and GD assisted in the plasmids constructing. CZ helped to analyze and interpreted the data. The others contributed to revision.

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Correspondence to Yankun Yang.

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Yang, Y., Pan, Y., Liu, G. et al. Glycerol transporter 1 (Gt1) and zinc-regulated transporter 1 (Zrt1) function in different modes for zinc homeostasis in Komagataella phaffii (Pichia pastoris). Biotechnol Lett 42, 2413–2423 (2020). https://doi.org/10.1007/s10529-020-02964-4

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Received: 01 February 2020
Accepted: 07 July 2020
Published: 13 July 2020
Issue Date: November 2020
DOI: https://doi.org/10.1007/s10529-020-02964-4

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Glycerol transporter 1 (Gt1) and zinc-regulated transporter 1 (Zrt1) function in different modes for zinc homeostasis in Komagataella phaffii (Pichia pastoris)