Effects of Arp2 and Arp3 nucleotide-binding pocket mutations on Arp2/3 complex function.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• The Arp2/3 complex is a key regulator of actin cytoskeleton dynamics, essential for cellular processes like endocytosis.
• The role of nucleotide binding by Arp2 and Arp3 subunits in complex function remains incompletely understood.

Purpose of the Study:

• To investigate the impact of nucleotide-binding pocket mutations in Arp2 and Arp3 on Arp2/3 complex activity in Saccharomyces cerevisiae.
• To elucidate the specific contributions of Arp2 and Arp3 nucleotide states to Arp2/3 complex function both in vitro and in vivo.

Main Methods:

• Construction and analysis of nucleotide-binding pocket (NBP) mutants in Arp2 and Arp3 in yeast.
• In vitro assays to measure Arp2/3 complex nucleation activity.
• Analysis of actin dynamics and endocytosis in mutant strains.
• Electron microscopy to visualize structural changes in the Arp2/3 complex.

Main Results:

• ATP binding by both Arp2 and Arp3 is required for maximal in vitro nucleation activity.
• Nucleotide-bound Arp3 is critical for Arp2/3 complex function in vivo, particularly for actin dynamics and endocytosis.
• Endocytic defects in mutants did not correlate with in vitro nucleation activity, suggesting a structural role.
• A distinct class of NBP mutants suppressed actin nucleation defects, with one Arp2 mutant enhancing nucleation and showing structural changes similar to activation.

Conclusions:

• Arp2 and Arp3 nucleotide binding are essential for Arp2/3 complex nucleating activity.
• Arp3 nucleotide binding is critical for maintaining the cortical actin cytoskeleton architecture.
• The Arp2/3 complex likely plays both catalytic and structural roles in cellular processes like endocytosis.