A temperature-sensitive splicing mutation in the bimG gene of Aspergillus produces an N-terminal fragment which interferes with type 1 protein phosphatase function.
Progression through anaphase requires high levels of type 1 protein phosphatase (PP1) activity in a variety of eukaryotes, including Aspergillus nidulans. A conditional lethal, temperature-sensitive mutant in one of the Aspergillus PP1 genes, bimG, prevents the normal completion of anaphase when cells are grown at restrictive temperature and this has been shown to be due to a reduction in type 1 phosphatase activity. We show that the bimG11 allele is recessive to the wild-type allele in heterozygous diploids, implying that the mutation is due to loss of function at restrictive temperature, but molecular disruption of the wild-type bimG gene shows that the gene is not essential and has no discernable phenotype under laboratory conditions. Sequence comparison of wild-type and mutant alleles reveals a single base pair difference between the two genes, within the 5' splicing site of the second intron. We demonstrate that the conditional lethal phenotype of bimG11 strains is due to impaired splicing of the mutant mRNA and that this leads to the production of a truncated protein comprising an intact N-subdomain and a modified C-terminus. Over-expression of this truncated form of PP1 in a wild-type haploid produces a lethal phenotype and reduced PP1 activity, supporting the idea that a toxic interfering protein is produced. PP1, therefore, may have at least two spatially separated sites, both of which are required for function. Temperature-sensitive splicing mutations may provide a novel means of engineering conditional versions of other proteins, particularly other phosphatases.
John Innes Centre, Norwich, UK.
M
M
Hughes
A
A
Arundhati
P
P
Lunness
P J
PJ
Shaw
J H
JH
Doonan
D000483:Alleles; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D001483:Base Sequence; D005800:Genes, Fungal; D005804:Genes, Lethal; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000235:genetics; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D013696:Temperature
D010455:Peptides; D010749:Phosphoprotein Phosphatases