Biomedical subjects

Elizabeth Vierling

Publications and source records attributed to Elizabeth Vierling.

22 records · Page 2Linked to original sources

PubMed2003

Arabidopsis UVH6, a homolog of human XPD and yeast RAD3 DNA repair genes, functions in DNA repair and is essential for plant growth.

To evaluate the genetic control of stress responses in Arabidopsis, we have analyzed a mutant (uvh6-1) that exhibits increased sensitivity to UV light, a yellow-green leaf coloration, and mild growth defects. We have mapped the uvh6-1 locus to chromosome I and have identified a candidate gene, AtXPD, within the corresponding region. This gene shows sequence similarity to the human (Homo sapiens) XPD and yeast (Saccharomyces cerevisiae) RAD3 genes required for nucleotide excision repair. We propose that UVH6 is equivalent to AtXPD because uvh6-1 mutants carry a mutation in a conserved residue of AtXPD and because transformation of uvh6-1 mutants with wild-type AtXPD DNA suppresses both UV sensitivity and other defective phenotypes. Furthermore, the UVH6/AtXPD protein appears to play a role in repair of UV photoproducts because the uvh6-1 mutant exhibits a moderate defect in the excision of UV photoproducts. This defect is also suppressed by transformation with UVH6/AtXPD DNA. We have further identified a T-DNA insertion in the UVH6/AtXPD gene (uvh6-2). Plants carrying homozygous insertions were not detected in analyses of progeny from plants heterozygous for the insertion. Thus, homozygous insertions appear to be lethal. We conclude that the UVH6/AtXPD gene is required for UV resistance and is an essential gene in Arabidopsis.

Adenosine Triphosphatases↗

PubMed2002

Small heat-shock proteins regulate membrane lipid polymorphism.

Thermal stress in living cells produces multiple changes that ultimately affect membrane structure and function. We report that two members of the family of small heat-shock proteins (sHsp) (alpha-crystallin and Synechocystis HSP17) have stabilizing effects on model membranes formed of synthetic and cyanobacterial lipids. In anionic membranes of dimyristoylphosphatidylglycerol and dimyristoylphosphatidylserine, both HSP17 and alpha-crystallin strongly stabilize the liquid-crystalline state. Evidence from infrared spectroscopy indicates that lipid/sHsp interactions are mediated by the polar headgroup region and that the proteins strongly affect the hydrophobic core. In membranes composed of the nonbilayer lipid dielaidoylphosphatidylethanolamine, both HSP17 and alpha-crystallin inhibit the formation of inverted hexagonal structure and stabilize the bilayer liquid-crystalline state, suggesting that sHsps can modulate membrane lipid polymorphism. In membranes composed of monogalactosyldiacylglycerol and phosphatidylglycerol (both enriched with unsaturated fatty acids) isolated from Synechocystis thylakoids, HSP17 and alpha-crystallin increase the molecular order in the fluid-like state. The data show that the nature of sHsp/membrane interactions depends on the lipid composition and extent of lipid unsaturation, and that sHsps can regulate membrane fluidity. We infer from these results that the association between sHsps and membranes may constitute a general mechanism that preserves membrane integrity during thermal fluctuations.

Feedback↗

PubMed2002

Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro.

The ability of small heat shock proteins (sHSPs) to prevent thermal aggregation of other proteins may require disassembly and reassembly of sHSP oligomers. We investigated the role of changes in sHSP oligomerization by studying a mutant with reduced oligomeric stability. In HSP16.6, the single sHSP in the cyanobacterium Synechocystis sp. PCC 6803, the mutation L66A causes oligomer instability and reduced chaperone activity in vitro. Because thermotolerance of Synechocystis depends on HSP16.6, a phenotype that is enhanced in a deltaClpB1 strain, the effect of mutations can also be assayed in vivo. L66A causes severe defects in thermotolerance, suggesting that oligomeric stability of sHSPs is required for cellular function. This hypothesis was supported by a selection for intragenic suppressors of L66A, which identified mutations that stabilize oligomers of both L66A and wild-type HSP16.6. Analysis of both over- and under-oligomerizing mutants suggests that sHSPs must disassemble before they can release substrates. Furthermore, the suppressor mutations not only restore in vivo activity to L66A, they also ameliorate chaperone defects in vitro, and thus provide the first direct evidence for a chaperone function of an sHSP in cellular thermotolerance.

Biopolymers↗

PubMed2002

Subunit exchange of multimeric protein complexes. Real-time monitoring of subunit exchange between small heat shock proteins by using electrospray mass spectrometry.

The subunit exchange of the small heat shock proteins (sHSPs) PsHSP18.1 from pea and TaHSP16.9 from wheat has been monitored in real-time using nanoelectrospray mass spectrometry. By preserving the noncovalent interactions between subunits in the mass spectrometer, we show that these proteins are dodecameric. After mixing PsHSP18.1 and TaHSP16.9, a distribution of heterododecamers is formed. A comparison with spectra obtained from statistical modeling demonstrates that after equilibration the distribution of these heterocomplexes is governed by the starting ratio of the two components rather than an inherent preference for certain stoichiometries. This finding suggests that the two different sHSP subunits interact in a very similar manner. Following the kinetics of this reaction by mass spectrometry reveals that exchange proceeds via sequential incorporation of subunits with dimeric species being the principal units of exchange. Therefore, we conclude that sHSP complexes are in rapid dissociation/reassociation equilibria with suboligomeric forms. More generally, these experiments illustrate a powerful approach for the real-time analysis of the evolution of transient species and their relative populations during the subunit exchange of multimeric protein complexes.

Heat-Shock Proteins↗