The role of membrane contact sites during prospore membrane
formation in budding yeast
Hiroyuki TACHIKAWA
Graduate School of Agricultural and Life Sciences, The University of Tokyo
Report:Prospore membrane (PSM) formation in budding yeast is a good model for de novo
membrane formation in the cell. We showed that membrane contact sites (MCSs) are formed
between the endoplasmic reticulum (ER) and the PSM and probably regulate phospholipid
composition of the PSM and PSM extension.
Research aims
Recent studies have revealed that MCSs are formed between organelles ororganelles and the plasma membrane and are required for lipid exchange betweenmembranes. We have been studying PSM formation in budding yeast as a model system fornew biological membrane formation in the cell and have shown that an MCS protein, Vps13,
and its sporulation-specific interacting proteins, Spo71, and Spo73, colocalize on the PSM
and are required for PSM extension1) 2). Further, we have revealed that the PSM extension
defect in spo73∆ is suppressed by decreasing the level of phosphatidylinositol 4-phosphate(PI4P) in the PSM, indicating the importance of the regulation of lipid composition of thePSM. It is known that the level of PI4P in the plasma membrane is decreased by MCSs
formed by tether proteins5). Therefore we hypothesized that MCSs were also formed
between the ER and the PSM, and regulated the lipid composition of the PSM and thesubsequent PSM extension. In this study, we examined the existence of MCSs between theER and the PSM, and suggested the possible role of MCSs in PSM formation.
Methods
Tether proteins were C-terminally tagged with GFP or GFPEnvy and observed witha PSM marker Spo2051-91-mKate2 by fluorescence microscopy in wild-type, spo73∆, spo71∆,
and vps13∆ cells during sporulation.
For Split-GFP analysis, one of the tether proteins, Tcb3, was tagged with GFP
strand 11, and Dtr1, a transmembrane PSM protein, was tagged with GFP strand 1-10.
These fusion proteins were expressed with Spo2051-91-mKate2 during sporulation inwild-type cells and observed by fluorescence microscopy.

Results
To examine whether tether proteins involved in the formation of MCSs between theER and the plasma membrane were localized to the PSM, we observed their localization
with a PSM marker protein during sporulation in wild type cells. We showed that three of thetether proteins, Ist2, Tcb3, and Scs2, localized to the PSM, suggesting the formation ofMCSs between the ER and the PSM (Figure 1 left). We next observed these tether proteins inspo73∆, spo71∆, and vps13∆ cells. As expected, tether proteins did not localize to the PSM(Figure 1 right), indicating that their localization was dependent on Spo73, Spo71, andVps13.
To further confirm the existence of MCSs between the ER and the PSM, one of thetether proteins Tcb3, which is a membrane protein of the ER, and a transmembrane PSM
protein, Dtr1, were tested to determine whether they were in close proximity or not using
split-GFP analysis (Figure 2A). GFP fluorescence along the PSM was detected (Figure 2B),
indicating that they were in close proximity to each other and that MCSs were formed
between the ER and the PSM.
Conclusion
In this study, we showed that tether proteins localized to the PSM and formed MCSs
between the ER and the PSM, dependent on Spo73, Spo71, and Vps13. Taking previous
work into consideration, our data indicate that MCSs are involved in the control of the levelsof PI4P in the PSM, which is important for PSM extension. This study will contribute to theunderstanding of the mechanism of de novo membrane formation in the cell.
References
1) Tachikawa H, Bloecher A, Tatchell K, Neiman AM. (2001) Gip1p-Glc7p phosphatasecomplex regulates septin organization and spore wall formation. J. Cell Biol. 155:797-808.
2) Nakamura TS, et al. (2017) Dynamic localization of a yeast development-specific PP1complex during prospore membrane formation is dependent on multiple localization
signals and complex formation Mol. Biol. Cell 28: 3881-3895.
3) Park JS, Okumura Y, Tachikawa H, Neiman AM (2013) SPO71 encodes adevelopmental stage-specific partner for Vps13 in Saccharomyces cerevisiae. Eukaryot.
Cell 12: 1530-1537.
4) Okumura Y, et al. (2016) The Dysferlin domain-only protein, Spo73, is required forprospore membrane extension in Saccharomyces cerevisiae. mSphere 1: 00038-15.
5) Manford AG, et al. (2012) ER-to-plasma membrane tethering proteins regulate cellsignaling and ER morphology. Dev. Cell 23: 1129-1140.

Figure 1. Localization of tether proteins during PSM formation in wild-type and spo73∆
cells. Ist2, Tcb3, and Scs2 tagged with GFP or GFPEnvy were observed by fluorescencemicroscopy in wild-type and spo73∆ cells with a PSM marker during sporulation.
AB

Figure 2. Split-GFP analysis of MCSs between the ER and the PSM. A. Schematic diagramsof Split-GFP analysis. GFP fluorescence can be detected only when Tcb3 is in close
proximity with Dtr1. B. GFP fluorescence observed with the PSM marker during
sporulation.