Structure of V-ATPase from citrus fruit

Area of Science:

• Biochemistry
• Structural Biology
• Plant Science

Background:

• Vacuolar-type ATPases (V-ATPases) are essential proton pumps found in eukaryotic cells.
• Maintaining cellular pH is critical for various biological processes, including fruit development and storage.
• Citrus fruit vacuoles typically exhibit a low pH, crucial for flavor and preservation.

Purpose of the Study:

• To determine the structure and function of the V-ATPase from lemon fruit.
• To investigate the enzyme's capacity to establish proton gradients relevant to citrus vacuoles.
• To compare plant V-ATPase structure with orthologs from other species.

Main Methods:

• Affinity purification of endogenous V-ATPases from lemon fruit using the Legionella pneumophila effector SidK.
• Cryoelectron microscopy for structure determination of the V-ATPase in two rotational states.
• Biochemical analysis to determine the ATP:H+ ratio and maximum pH gradient (ΔpH).

Main Results:

• The V-ATPase structure was resolved in two distinct rotational states.
• The enzyme establishes a maximum ΔpH of approximately 3, which is insufficient for lemon vacuolar pH.
• Plant V-ATPase lacks subunit f and exhibits an unusual transmembrane helix configuration compared to yeast and mammalian counterparts.
• Subunit H displays two conformations, suggesting a regulatory role in enzyme activity.

Conclusions:

• The lemon V-ATPase structure provides insights into plant-specific enzyme architecture.
• The determined proton pumping capacity is insufficient to explain the low pH of citrus vacuoles.
• Plant V-ATPases possess unique structural features and regulatory mechanisms, such as subunit H conformations, potentially influencing their function in vivo.