Suppression of Vps13 adaptor protein mutants reveals a central role for PI4P in regulating prospore membrane
Tsuyoshi S Nakamura1,2, Yasuyuki Suda3,4, Kenji Muneshige1
1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Abstract:
Vps13 family proteins are proposed to function in bulk lipid transfer between membranes, but little is known about their regulation. During sporulation of Saccharomyces cerevisiae, Vps13 localizes to the prospore membrane (PSM) via the Spo71-Spo73 adaptor complex. We previously reported that loss of any of these proteins causes PSM extension and subsequent sporulation defects, yet their precise function remains unclear. Here, we performed a genetic screen and identified genes coding for a fragment of phosphatidylinositol (PI) 4-kinase catalytic subunit and PI 4-kinase noncatalytic subunit as multicopy suppressors of spo73Δ. Further genetic and cytological analyses revealed that lowering PI4P levels in the PSM rescues the spo73Δ defects. Furthermore, overexpression of VPS13 and lowering PI4P levels synergistically rescued the defect of a spo71Δ spo73Δ double mutant, suggesting that PI4P might regulate Vps13 function. In addition, we show that an N-terminal fragment of Vps13 has affinity for the endoplasmic reticulum (ER), and ER-plasma membrane (PM) tethers localize along the PSM in a manner dependent on Vps13 and the adaptor complex. These observations suggest that Vps13 and the adaptor complex recruit ER-PM tethers to ER-PSM contact sites. Our analysis revealed that involvement of a phosphoinositide, PI4P, in regulation of Vps13, and also suggest that distinct contact site proteins function cooperatively to promote de novo membrane formation.
Insights
Phosphatidylinositol 4-phosphate (PI4P) regulates Vps13 protein function in lipid transfer during yeast sporulation. Lowering PI4P levels rescues defects caused by Vps13 adaptor complex loss, aiding membrane formation.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Vps13 proteins are implicated in bulk lipid transfer between membranes.
- Their regulatory mechanisms and precise functions remain largely unknown.
- Vps13 localizes to the prospore membrane (PSM) during yeast sporulation via the Spo71-Spo73 adaptor complex.
Purpose of the Study:
- To elucidate the regulation of Vps13 function during Saccharomyces cerevisiae sporulation.
- To investigate the role of phosphatidylinositol 4-phosphate (PI4P) in Vps13-mediated processes.
- To understand the recruitment of ER-PM tethers to ER-PSM contact sites.
Main Methods:
- Genetic screening to identify suppressors of spo73Δ mutants.
- Cytological analyses to assess PSM morphology and protein localization.
- Investigating the interaction between Vps13, the adaptor complex, PI4P levels, and ER-PM tethers.
Main Results:
- Genes encoding fragments of PI 4-kinase subunits were identified as multicopy suppressors of spo73Δ.
- Reduced PI4P levels in the PSM rescued defects associated with spo73Δ.
- Overexpression of VPS13 and reduced PI4P synergistically rescued spo71Δ spo73Δ double mutant defects.
- Vps13 recruits ER-PM tethers to ER-PSM contact sites in a Vps13 and adaptor complex-dependent manner.
Conclusions:
- Phosphatidylinositol 4-phosphate (PI4P) plays a regulatory role in Vps13 function.
- The Vps13-adaptor complex recruits ER-PM tethers to facilitate de novo membrane formation at ER-PSM contact sites.
- Distinct contact site proteins cooperate to promote membrane biogenesis during sporulation.
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