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SCIENTIA SINICA Vitae, Volume 49, Issue 9: 1165-1172(2019) https://doi.org/10.1360/SSV-2019-0155

The endoplasmic reticulum-plasma membrane tethering protein Ist2 regulates endoplasmic reticulum stress response in Candida albicans

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  • ReceivedJul 22, 2019
  • AcceptedAug 12, 2019
  • PublishedSep 9, 2019

Abstract

The endoplasmic reticulum-plasma membrane contact (ER-PM contact) is a novel membrane contact system in eukaryotic cells that mediates the close exchange of materials and messengers between ER and PM. However, the types and function of the ER-PM tethering proteins of the opportunistic pathogenic fungus Candida albicans remain poorly understood. In the present study, we found that Ist2 co-localizes with the ER-PM contact sites of C. albicans and that its deletion causes a marked decrease in the ER-PM contact sites as revealed by fluorescence observation. This indicates that Ist2 is a key ER-PM tethering protein of C. albicans. The measurement of growth curves, fluorescence detection of the unfolded protein response (UPR) reporting system, and the measurement of the cytosolic Ca2+ level revealed that the IST2 deletion strain ist2Δ/Δ considerably increased in biomass, the UPR reporting gene PRB1 expression was attenuated, and the cytoplasmic Ca2+ levels under ER stress caused by tunicamycin decreased. In addition, the Ca2+ chelating agent EGTA could eliminate the difference between ist2Δ/Δ and WT in ER stress tolerance. Therefore, Ist2 is an important ER-PM tethering protein in C. albicans and a negative regulator of ER stress responses, which mediates an increase in cytosolic Ca2+ level under ER stress, resulting in attenuated ER stress tolerance. The results of the present study facilitate our understanding of the structure and function of the fungal ER-PM contact and provide an important basis for the development of novel antifungal targets.


Funded by

国家自然科学基金(31870139,81873961)

天津市自然科学基金(17JCZDJC33300)

中央高校基本科研业务费


References

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  • Figure 1

    Localization of Ist2 in Candida albicans. A: Co-localization of Ist2-GFP and the ER indicator Sec61-RFP (indicated by arrows); B: Co-localization of Ist2-GFP and the PM indicator PH3-RFP (indicated by arrows). Cells were also stained using DAPI to indicate the nuclei. Scale bar=5 μm

  • Figure 2

    Morphological observation of ER-PM contact sites in WT and ist2Δ/Δ strains (indicated by arrows). Scale bar= 5 μm

  • Figure 3

    Effect of IST2 deletion on the growth curves of C. albicans under normal (A) and ER stress (B) conditions. A: The growth curves of WT and ist2Δ/Δ under the normal YPD culturing condition. B: The growth curves of the strains in YPD medium containing 4-μg/mL TN. “*” indicates significant difference in A600 nm between WT and ist2Δ/Δ (P<0.05)

  • Figure 4

    Expression of PRB1 in WT and ist2Δ/Δ strains under normal and 4-μg/mL TN-treatment conditions. “*” Indicates significant differences between the groups (P<0.05)

  • Figure 5

    Effect of IST2 deletion on cytoplasmic Ca2+ contents in WT and ist2Δ/Δ strains under normal and TN-treatment conditions. “*” Indicates significant differences between the groups (P<0.05)

  • Figure 6

    Effect of EGTA on cell growth (A) and intracellular PRB1 expression (B). A: The strains were treated using 4 μg/mL TN and 1 mmol/L EGTA alone or in combination; then the A600 nm of cultures was measured to reveal biomass growth. B: The fluorescence intensity of the strains containing the PPRB1-GFP reporting system under treatment with TN, EGTA, or TN+EGTA. “*” Indicates significant differences between the groups, and “ns” indicates no significant difference between the groups (P<0.05)

  • Table 1   Strains used in the present study

    菌株

    基因型

    来源

    WT

    (野生型菌株)

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG

    本实验室保存

    ist2Δ/Δ

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG ist2::ARG4/ist2::URA3-dpl200

    本研究构建

    WT-GFP-Ist2-Sec61-RFP

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG act1:: PACT1-GFP-IST2-URA3 act1:: PACT1-SEC61-RFP-URA3

    本研究构建

    WT-GFP-Ist2-PH3-RFP

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG act1:: PACT1-GFP-IST2-URA3 act1:: PACT1-PH3-RFP-URA3

    本研究构建

    WT-Sec61-GFP-PH3-RFP

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG act1:: PACT1-SEC61-GFP-URA3 act1:: PACT1-PH3-RFP-URA3

    本研究构建

    ist2Δ/Δ-Sec61-GFP-PH3-RFP

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG ist2::ARG4/ist2::URA3-dpl200 act1:: PACT1-SEC61-GFP-URA3 act1:: PACT1-PH3-RFP-URA3

    本研究构建

    WT-PPRB1-GFP

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG pPPRB1-GFP

    本研究构建

    ist2Δ/Δ- PPRB1-GFP

    ura3Δ::λimm434/ura3Δ::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG ist2::ARG4/ist2::URA3-dpl200 pPPRB1-GFP

    本研究构建

  • Table 2   Plasmids used in the present study

    质粒

    性质

    来源

    pRS-ArgΔSpe І

    ARG4 selectable marker, AmpR

    本实验室保存

    pDDB57

    URA3 selectable marker, AmpR

    本实验室保存

    pDDB78

    URA3 & TRP1 selectable marker, AmpR

    本实验室保存

    pAU34M-GFP-Ist2

    URA3 selectable marker, AmpR

    本实验室保存

    pAU34M-Sec61-GFP

    URA3 selectable marker, AmpR

    本实验室保存

    PDDB78-PH3-RFP

    HIS1 selectable marker, AmpR

    本实验室保存

    pPPRB1-GFP

    URA3 selectable marker, pPPRB1-GFP, AmpR

    本实验室保存

  • Table 3   Primers used in the present study

    引物

    序列(5′→3′)

    作用

    IST2-5DR

    TAATTTATTACTTATTCAATATATTTTTTTTCTTCCTTCCCTTCTTCAACTTCTTCTTTATTTCCCAGTCACGACGTT

    敲除IST2基因(画线部分为筛选标记PCR用序列)

    IST2-3DR

    TCATCATTATTTTCAACTTTATTGACAGCATTGGTAACTAATTTTTTAGGAGATGTTTTTTGGAATTGTGAGCGGATA

    IST2-5det

    GTTTGCTTGTTTGCTCATATTTAC

    验证IST2敲除菌株

    IST2-3det

    TTCATACAACTTATATCGAGACAATT

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