PKR1 encodes an assembly factor for the yeast V-type ATPase

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The V-ATPase is a crucial proton pump involved in various cellular functions, including vacuolar acidification and metal ion transport.
• Efficient assembly of the V-ATPase complex in the endoplasmic reticulum (ER) is critical for its function.
• Iron transport in yeast relies on high-affinity systems involving proteins like Fet3p, which require proper V-ATPase function.

Purpose of the Study:

• To investigate the role of the yeast gene PKR1 (YMR123W) in V-ATPase assembly and function.
• To elucidate the mechanism by which Pkr1p influences V-ATPase subunit stability and cellular iron uptake.
• To determine if Pkr1p acts as a V-ATPase assembly factor.

Main Methods:

• Gene deletion (pkr1Δ) in Saccharomyces cerevisiae.
• Analysis of V-ATPase subunit (Vph1p) levels and turnover.
• Assessment of Fet3p copper loading and high-affinity iron transport.
• Vacuolar acidification assays.
• Complementation studies using V-ATPase assembly factors (Vma21p).

Main Results:

• Deletion of PKR1 resulted in impaired growth on iron-limited medium.
• Cells lacking Pkr1p exhibited reduced levels of the V-ATPase subunit Vph1p due to increased protein turnover.
• Defective V-ATPase function in pkr1Δ cells led to impaired copper loading of Fet3p.
• Despite low Vph1p levels, the assembled V-ATPase in pkr1Δ cells was functional, as indicated by weak vacuolar acidification.
• Overexpression of Vma21p suppressed the growth and acidification defects of pkr1Δ cells.

Conclusions:

• Pkr1p is a novel factor involved in the efficient assembly of the V-ATPase membrane sector within the ER.
• Pkr1p likely functions in conjunction with other known V-ATPase assembly factors.
• The role of Pkr1p in V-ATPase assembly directly impacts cellular iron homeostasis.