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The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane

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Abstract

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is generated through phosphorylation of phosphatidylinositol 4-phosphate (PtdIns(4)P) by Mss4p, the only PtdIns phosphate 5-kinase in yeast cells. PtdIns(4,5)P2 is involved in various kinds of yeast functions. PtdIns(4)P is not only the immediate precursor of PtdIns(4,5)P2, but also an essential signaling molecule in the plasma membrane, Golgi, and endosomal system. To analyze the distribution of PtdIns(4,5)P2 and PtdIns(4)P in the yeast plasma membrane at a nanoscale level, we employed a freeze–fracture electron microscopy (EM) method that physically immobilizes lipid molecules in situ. It has been reported that the plasma membrane of budding yeast can be divided into three distinct areas: furrowed, hexagonal, and undifferentiated flat. Previously, using the freeze–fracture EM method, we determined that PtdIns(4)P is localized in the undifferentiated flat area, avoiding the furrowed and hexagonal areas of the plasma membrane. In the present study, we found that PtdIns(4,5)P2 was localized in the cytoplasmic leaflet of the plasma membrane, and concentrated in the furrowed area. There are three types of PtdIns 4-kinases which are encoded by stt4, pik1, and lsb6. The labeling density of PtdIns(4)P in the plasma membrane significantly decreased in both pik1ts and stt4ts mutants. However, the labeling densities of PtdIns(4,5)P2 in the plasma membrane of both the pik1ts and stt4ts mutants were comparable to that of the wild type yeast. These results suggest that PtdIns(4)P produced by either Pik1p or Stt4p is immediately phosphorylated by Mss4p and converted to PtdIns(4,5)P2 at the plasma membrane.

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Abbreviations

GFP:

Green fluorescent protein

PLC:

Phospholipase C

BSA:

Bovine serum albumin

EF:

E-face

EM:

Electron microscopy

IMP:

Intramembrane particles

PBS:

Phosphate-buffered saline

PF:

P-face

QF:

Quick freezing

SDS:

Sodium dodecyl sulfate

WT:

Wild type

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Acknowledgements

We thank Dr. Toyoshi Fujimoto (Juntendo University) for the kind gift of the PH-PLC-δ1/pGEX plasmid.

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS); Grant-in-Aid for Scientific Research (KAKENHI) [grant numbers JP17H03935, JP16K15056, 19K22435, and 20H03154]; and research grants from Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering, Takeda Science Foundation, The Naito Foundation, ONO Medical Research Foundation, The NOVARTIS Foundation (Japan) for the Promotion of Science, and The Uehara Memorial Foundation (to A.F.).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology and Biochemistry, Basic Veterinary Medicine, Faculty of Veterinary Medicine, Kagoshima University, Korimoto 1-21-24, Kagoshima, 890-0065, Japan

    Yuna Kurokawa, Rikako Konishi, Kanna Tomioku & Akikazu Fujita

  2. Medical Research Institute, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo, 162-8666, Japan

    Kenji Tanabe

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  1. Yuna Kurokawa
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  2. Rikako Konishi
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  3. Kanna Tomioku
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  5. Akikazu Fujita
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Correspondence to Akikazu Fujita.

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418_2021_1989_MOESM1_ESM.tiff

Supplementary file1 Fig. S1. The labeling densities of PtdIns(4)P in the furrowed, hexagonal, and flat areas. The labeling density of PtdIns(4)P in the flat undifferentiated membrane was denser than in hexagonal and furrowed areas. (TIFF 1956 KB)

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Kurokawa, Y., Konishi, R., Tomioku, K. et al. The distribution of phosphatidylinositol 4,5-bisphosphate in the budding yeast plasma membrane. Histochem Cell Biol 156, 109–121 (2021). https://doi.org/10.1007/s00418-021-01989-8

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