Key result
Muscle/yeast hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, mitochondrial defects, and altered polymerization dynamics, demonstrating yeast sensitivity to actin N-terminal negative charge density.
The study demonstrates a unique sensitivity of yeast to actin N-terminal negative charge density and provides insight into the role of specific domains in actin function.
Yeast findings remain preclinical without human applicability; leaves open actin N-terminal charge as a regulator of cytoskeletal function.
Muscle and yeast actins display distinct behavioral characteristics. To better understand the allosteric interactions that regulate actin function, we created a muscle/yeast hybrid actin containing a muscle-specific outer domain (subdomains 1 and 2) and a yeast inner domain (subdomains 3 and 4). Actin with muscle subdomain 1 and the two yeast N-terminal negative charges supported viability. The four negative charge muscle N terminus in a muscle subdomain 1 background caused death, but in the same background actin with three N-terminal acidic residues (3Ac/Sub1) led to sick but viable cells. Addition of three muscle subdomain 2 residues (3Ac/Sub12) produced no further deleterious effects. These hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, and mitochondrial and endocytosis defects. 3Ac/Sub1 G-actin exchanged bound epsilonATP more slowly than wild type actin, and the exchange rate for 3Ac/Sub12 was even slower, similar to that for muscle actin. The mutant actins polymerized faster and produced less stable and shorter filaments than yeast actin, the opposite of that expected for muscle actin. Unlike wild type actin, in the absence of unbound ATP, polymerization led to ADP-F-actin, which rapidly depolymerized. Like yeast actin, the hybrid actins activated muscle myosin S1 ATPase activity only about one-eighth as well as muscle actin, despite having essentially a muscle actin-specific myosin-binding site. Finally, the hybrid actins behaved abnormally in a yeast Arp2/3-dependent polymerization assay. Our results demonstrate a unique sensitivity of yeast to actin N-terminal negative charge density. They also provide insight into the role of each domain in the control of the various functions of actin.
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McKane et al. (2006) studied this question. Muscle/yeast hybrid actin vs. Wild type yeast actin and muscle actin was evaluated on Actin function (viability, polymerization, ATPase activity). Muscle/yeast hybrid actins caused depolarized cytoskeletons, abnormal vacuoles, mitochondrial defects, and altered polymerization dynamics, demonstrating yeast sensitivity to actin N-terminal negative charge density.
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