The Effects of Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop on the Dynamics and Stability of Yeast G-actin

Abstract

The hydrophobicity of the subdomain 3/4 hydrophobic loop (262-274) has been implicated to be essential for actin's function. We previously showed (Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, 1237-1247) that a mutant yeast actin (V266G/L267G) with markedly decreased hydrophobicity in this loop conferred severe cold sensitivity to its polymerization. Here we further tested the mutational effect on the conformation and function of G-actin. This GG mutation caused no significant changes in overall secondary structure or in the microenvironment around actin's tryptophan residues, nor did it alter the dissociation constant of G-actin for ATP. However, it lowers the intrinsic ATPase activity and the melting temperature for Mg-GG actin from 51 to 33°C and transforms the conformation of subdomain 2 and the central cleft of G-actin into an F-monomer-like structure. The results suggest that the hydrophobic plug may not only play a role in actin filament stabilization but also may be important for controlling the stability of G-actin and for promoting the conformational change of the monomer needed for addition to a growing actin filament.

Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization

“…The original Holmes model for F-actin involved a postulated ''hydrophobic plug'' between the two strands, with a loop (residues 263-274) that swung out from the body of the monomer to make a contact with subunits on the opposing strand (20). Subsequent experiments have partially supported this model (21)(22)(23), and it has been shown that locking this loop to the body of the actin subunit by engineered disulfide bonds can prevent normal polymerization (16). We have used the yeast actin triple mutant (L180C͞L269C͞C374A), which places two cysteine residues in positions allowing for the loop to be locked to the body of actin and eliminates the reactive cysteine at position 374 (16).…”

A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype

“…Pertinent to this proposition is the recognition that in yeast mitochondrial integrity depends on a carefully regulated fission/fusion cycle which, when interrupted, can result in mitochondrial abnormalities and a loss of mtDNA (43). Hill et al (22) showed that one of the alanine scanning mutants, act1-101, had both a mitochondrial and a vacuolar inheritance phenotype (42). We have recently observed that in this cell the vacuole is also hypervesiculated (data not shown).…”

“…Initially, an assessment of vacuole integrity and inheritance was not part of the characterization of the alaninescanning actin mutants. However, other studies revealed, using yeast actin mutations generated by us (22,42), that actin was involved in both vacuole morphology determination and vacuole inheritance and that mutations could be found that affected vacuole behavior independent of mitochondria (41).…”

Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin–actin interactions in budding yeast

“…WT yeast actin (a generous gift from P. Rubenstein, University of Iowa, Iowa City) was purified by a DNase I affinity chromatography protocol as described (31,32). F-actin sedimentation assays were performed as described (33).…”