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Abstract
We have examined the role of the acidic residues Asp2 and Glu4 at the NH2 terminus of Saccharomyces cerevisiae actin through site-directed mutagenesis. In DNEQ actin, these residues have been changed to Asn2 and Gln4, whereas in delta DSE actin, the Asp2-Ser-Glu tripeptide has been deleted. Both mutant actins can replace wild type yeast actin. Peptide mapping studies reveal that DNEQ, like wild type actin, retains the initiator Met and is NH2 terminally acetylated, whereas delta DSE has a free NH2 terminus and has lost the initiator Met. Interestingly, microscopic examination of filaments of these two actins reveal the appearance of bundled filaments. The DNEQ bundles are smaller and more ordered, whereas the delta DSE bundles are larger and more loosely organized. Additionally, both mutant actins activate the ATPase activity of rabbit muscle myosin S1 fragment to a lesser extent than wild type. We have also developed a sensitive assay for actin function in vivo that enabled us to detect a slight defect in the ability of these mutant actins to support secretion, an important function in yeast. Thus, although the mutant actins resulted in no gross phenotypic changes, we were able to detect a defect in actin function through this assay. From these studies we can conclude that 1) although NH2-terminal negative charges are not essential to yeast life, the loss of such charges does result in a slight defect in the actins' ability to support secretion, 2) removal of the NH2-terminal negative charges promotes the bundling of actin filaments, and 3) actins lacking NH2-terminal negative charges are unable to activate the myosin S1 ATPase activity as well as wild type actin.
Highlights
We have examined the role of the acidic residues Asp’ and Glu‘ at the NHz terminus of Saccharomyces cerevisiae actin through site-directedmutagenesis
The DNEQ bundles are smaller andmore ordered, whereas theADSE bundles cross-linking is unlikely to occur [13], it has been demonstrated that these amino-terminal acidicresidues arethe acidic residuesmost likely to react with l-ethyl-3-(3-dimethyaminopropy1)carbodiimide[14].Site-directedmutagenesisof these acidic residues is a way in which to directly test their role in actin function without concern for their general reactivity to cross-linking agents
At least 1 acidic residue remained at the actin NH2 terminus, and in one case a bulky positively charged imidazolewas substituted for the acidic residues.This substiterminal negative charges promotes the bundling of tution might possibly cause steric hindrance of the proteinactin filaments, and 3) actins lacking NHz-terminal protein interaction, leading to the observed effect
Summary
Strom andKarlsson [16]examined a series of mutant chicken bothmutantactinsactivatethe ATPase activity of @-actinsexpressed in yeast In these experiments, 2 of the 3 rabbit muscle myosin S1 fragment to a lesser extent NHZ-terminalAsp residues were either deleted or changed to than wild type. The mutant actins resulted inno gross phenotypic changes, we were able to detecatdefect in actin function through this assay From these studies we can conclude that 1) NHz-terminal negative charges are not essential to yeast life, the loss of such charges does result ina slight defect in theactins’ ability to support secretion, 2) removal of the NHz-. Assays of secreted invertase were as described [28]
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