DOI: 10.1091/mbc.11.4.1345. A Screen for Dominant Negative Mutants of<i>SEC18</i>Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

A Screen for Dominant Negative Mutants ofSEC18Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis

10.1091/mbc.11.4.1345

Crossref journal-article

American Society for Cell Biology (ASCB)

Molecular Biology of the Cell (1076)

Abstract

An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum–Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex ofN-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure–function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulatedSEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. Thesec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr394→Pro). Thr394is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast α-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts.

Bibliography

Steel, G. J., Harley, C., Boyd, A., & Morgan, A. (2000). A Screen for Dominant Negative Mutants ofSEC18Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis. Molecular Biology of the Cell, 11(4), 1345â1356.

Authors 4

Gregor J. Steel (first)
Carol Harley (additional)
Alan Boyd (additional)
Alan Morgan (additional)

References 42 Referenced 16

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Dates

Type	When
Created	12 years, 1 month ago (July 2, 2013, 7:50 p.m.)
Deposited	3 years, 6 months ago (Feb. 26, 2022, 9:41 p.m.)
Indexed	1 year ago (Aug. 5, 2024, 12:36 a.m.)
Issued	25 years, 4 months ago (April 1, 2000)
Published	25 years, 4 months ago (April 1, 2000)
Published Print	25 years, 4 months ago (April 1, 2000)

Funders 0

None

BibTeX

@article{Steel_2000, title={A Screen for Dominant Negative Mutants ofSEC18Reveals a Role for the AAA Protein Consensus Sequence in ATP Hydrolysis}, volume={11}, ISSN={1939-4586}, url={http://dx.doi.org/10.1091/mbc.11.4.1345}, DOI={10.1091/mbc.11.4.1345}, number={4}, journal={Molecular Biology of the Cell}, publisher={American Society for Cell Biology (ASCB)}, author={Steel, Gregor J. and Harley, Carol and Boyd, Alan and Morgan, Alan}, editor={Walter, Peter}, year={2000}, month=apr, pages={1345–1356} }

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  "abstract": "<jats:p>An evolutionarily ancient mechanism is used for intracellular membrane fusion events ranging from endoplasmic reticulum\u2013Golgi traffic in yeast to synaptic vesicle exocytosis in the human brain. At the heart of this mechanism is the core complex ofN-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). Although these proteins are accepted as key players in vesicular traffic, their molecular mechanisms of action remain unclear. To illuminate important structure\u2013function relationships in NSF, a screen for dominant negative mutants of yeast NSF (Sec18p) was undertaken. This involved random mutagenesis of a GAL1-regulatedSEC18 yeast expression plasmid. Several dominant negative alleles were identified on the basis of galactose-inducible growth arrest, of which one, sec18-109, was characterized in detail. Thesec18-109 phenotype (abnormal membrane trafficking through the biosynthetic pathway, accumulation of a membranous tubular network, growth suppression, increased cell density) is due to a single A-G substitution in SEC18 resulting in a missense mutation in Sec18p (Thr<jats:sub>394</jats:sub>\u2192Pro). Thr<jats:sub>394</jats:sub>is conserved in most AAA proteins and indeed forms part of the minimal AAA consensus sequence that serves as a signature of this large protein family. Analysis of recombinant Sec18-109p indicates that the mutation does not prevent hexamerization or interaction with yeast \u03b1-SNAP (Sec17p), but instead results in undetectable ATPase activity that cannot be stimulated by Sec17p. This suggests a role for the AAA protein consensus sequence in regulating ATP hydrolysis. Furthermore, this approach of screening for dominant negative mutants in yeast can be applied to other conserved proteins so as to highlight important functional domains in their mammalian counterparts.</jats:p>",
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    {
      "given": "Peter",
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}