Functional complementation in yeast allows molecular characterization of missense argininosuccinate lyase mutations.
Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL(null) strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, "compound heterozygous" yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.
Trevisson
Eva
E
Clinical Genetics Unit, University of Padova, 35128 Padova, Italy.
Burlina
Alberto
A
Doimo
Mara
M
Pertegato
Vanessa
V
Casarin
Alberto
A
Cesaro
Luca
L
Navas
Placido
P
Basso
Giuseppe
G
Sartori
Geppo
G
Salviati
Leonardo
L
D000483:Alleles; D001123:Argininosuccinate Lyase / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005838:Genotype; D006238:Haploidy; D006579:Heterozygote; D006801:Humans; D008957:Models, Genetic; D008968:Molecular Conformation; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014508:Urea / Q000378:metabolism
D014508:Urea; D001123:Argininosuccinate Lyase
Hum Mutat. 2007 Jul;28(7):694-702
17326097
Proc Natl Acad Sci U S A. 1984 Jul;81(14):4480-4
6589607
Nat Rev Genet. 2006 Jun;7(6):449-60
16708072
J Inherit Metab Dis. 2002 Sep;25(5):399-410
12408190
Hum Mol Genet. 2000 May 1;9(8):1245-9
10767350
Anal Biochem. 1990 Dec;191(2):384-9
2085183
J Biol Chem. 1991 Mar 15;266(8):5286-90
1705937
Biochem Biophys Res Commun. 2006 Mar 17;341(3):810-5
16438935
J Biol Chem. 1997 Mar 7;272(10):6777-83
9045711
J Inherit Metab Dis. 2000 Jun;23(4):308-12
10896281
Proc Natl Acad Sci U S A. 1990 Dec;87(24):9625-9
2263616
Nature. 1970 Aug 15;227(5259):680-5
5432063
J Clin Invest. 2007 Mar;117(3):765-72
17332895
J Biol Chem. 2002 Mar 29;277(13):11473-80
11790793
Hum Genet. 2002 Oct;111(4-5):350-9
12384776
Methods Mol Biol. 2006;313:107-20
16118429
Biochemistry. 2001 Dec 25;40(51):15570-80
11747432
Proc Natl Acad Sci U S A. 1997 Aug 19;94(17):9063-8
9256435
Hum Mol Genet. 2008 Jan 15;17(2):201-14
17925330
Yeast. 1991 Aug-Sep;7(6):609-15
1767589
Proc Natl Acad Sci U S A. 1986 Oct;83(19):7211-5
3463959
J Biol Chem. 2004 Sep 24;279(39):40972-9
15273245