Elizabeth Vierling - Academia.edu (original) (raw)

Papers by Elizabeth Vierling

[Advanced Search][Browse the Archive] Institution: UNIV OF MASSACHUSETTS, Amherst Sign In Adverti... more [Advanced Search][Browse the Archive] Institution: UNIV OF MASSACHUSETTS, Amherst Sign In Advertisement 10,000 Expression ORF Clones for $98 each Advertisement JBC\u27s Young Investigator Award: Learn More Mutants in a Small Heat Shock Protein That Affect the Oligomeric State ANALYSIS AND ALLELE-SPECIFIC SUPPRESSION* Kim C. Giese and Elizabeth Vierling‡ + Author Affiliations Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721 ‡ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biophysics, University of Arizona, 1007 E. Lowell St., Tucson, AZ 85721. Tel.: 520-621-1601; Fax: 520-621-3709; E-mail: vierling@email.arizona.edu. Next Section Abstract Oligomerization is an essential property of small heat shock proteins (sHSPs) that appears to regulate their chaperone activity. We have examined the role of conserved hydrophobic residues that are postulated to stabilize sHSP oligomers. We identified a mutation of Synechocystis Hsp16.6 that impairs function in vivo and in vitro. The V143A mutation is in the C-terminal extension, a region predicted to form an oligomeric interaction with a hydrophobic region that includes the site of a previously characterized mutation, L66A. Both mutants were dimeric, but V143A had a stronger oligomerization defect than L66A. However, V143A protected a model substrate better than L66A. This suggests that although the two regions both play a role in oligomerization, they are not equivalent. Nevertheless, the addition of either dimeric sHSP enhanced the in vitro chaperone activity of wild type Hsp16.6, consistent with models that the sHSP dimers initiate interactions with substrates. Suppressor analysis of V143A identified mutations in the N terminus that restored activity by restabilizing the oligomer. These mutants were allele-specific and unable to suppress L66A, although they suppressed a dimeric C-terminal truncation of Hsp16.6. Conversely, suppressors of L66A were unable to suppress either V143A or the truncation, although they, like suppressors of V143A, stabilize the Hsp16.6 oligomer. We interpret these data as evidence that the mutations V143A and L66A stabilize two different dimeric structures and as further support that sHSP dimers are active species. Previous SectionNext Section Small heat shock proteins (sHSPs)1 are a family of ATP-independent chaperones that, in vitro, can protect denaturing proteins from irreversible aggregation by forming large, soluble complexes with their substrates (for reviews, see Refs. 1-3). sHSPs do not refold substrates, but sHSP-protected substrates can be refolded by ATP-dependent chaperones such as Hsp70/DnaK (4, 5). Although the role of sHSPs in vivo is not well defined, established in vitro assays for sHSP activity (i.e. protection of model substrates from aggregation and their subsequent refolding by ATP-dependent chaperones) appear to be closely related to sHSP functions in vivo. In Synechocystis sp. strain PCC 6803 (Synechocystis), a missense mutation of the single sHSP in this organism, Hsp16.6, causes loss of both protection of a model substrate in vitro and the ability of cells to survive heat treatment (thermotolerance). Both activities are restored by intragenic suppressors of this mutant (6). Mogk et al. (7) demonstrated that the sHSPs in Escherichia coli act with both DnaK and ClpB to facilitate the removal of aggregated protein after heat stress, in good agreement with in vitro observations of Hsp70/DnaK-dependent reactivation of sHSP-protected substrates (8, 9). Thus, it appears that sHSPs function to prevent or reduce irreversible protein aggregation during heat stress in vivo. The mechanism of the chaperone activity of sHSPs is unknown. Current models suggest that changes in sHSP oligomerization are essential. Almost all sHSPs form oligomers (2), and these can be destabilized at high temperatures (10, 11). Disassembly of the oligomer into dimers has been proposed to be a required step in the chaperone process (10). Disassembly could produce an active state of the sHSP that can bind unfolding proteins (10), perhaps by exposure of hydrophobic residues that bind substrates (5, 11, 12). However, the oligomeric state is also essential, as it appears that reassembly of sHSP dimers into a complex with substrate is required for protection of substrates (6, 13-16). Therefore, identification of contacts involved in oligomerization is important for understanding the mechanism of sHSP chaperone activity. The crystal structures of two sHSPs provide information about regions involved in oligomerization. The structures of Methanococcus jannaschii Hsp16.5 (17) and Triticum aestivum Hsp16.9 from wheat (11) reveal that despite the difference in the size of their oligomers (24 and 12 subunits, respectively), there is a high degree of similarity between the two sHSPs. Both proteins form oligomers that are composed of dimers, and the conserved ∼100 amino acid α-crystallin…

Frontiers in Plant Science, 2021

Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher or... more Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form S-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme S-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an Arabidopsis thaliana GSNOR null mutant (hot5-2/gsnor1-3). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in hot5-2/gsnor1-3 plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8...

Summary. A low molecular weight heat shock protein which localizes to chloroplasts has been ident... more Summary. A low molecular weight heat shock protein which localizes to chloroplasts has been identified in several plant species. This protein belongs to a eukaryotic superfamily of small HSPs, all of which contain a conserved carboxyl-terminal domain. To investigate further the structure of this HSP, we isolated and sequenced cDNA clones for the chloroplast LMW HSPs from Petunia hybrida and Arabidopsis thaliana. The cloning of chloroplast HSPs from these two species enabled us to compare the amino acid sequences of this protein from plant species (petunia, Arabidopsis, pea, soybean and maize) that represent evolutionarily divergent taxonomic subclasses. Three conserved regions were identified, which are designated as regions I, II and III. Regions I and II are also shared by cytoplasmic LMW HSPs and therefore are likely to have functional roles common to all eukaryotic LMW HSPs. In contrast, consensus region III is not found in other LMW HSPs. Secondary structure analysis predicts t...

New Phytologist, 2021

The nucellus tissue in flowering plants provides nutrition for the development of the female game... more The nucellus tissue in flowering plants provides nutrition for the development of the female gametophyte (FG) and young embryo. The nucellus degenerates as the FG develops, but the mechanism controlling the coupled process of nucellar degeneration and FG expansion remains largely unknown. The degeneration process of the nucellus and spatiotemporal auxin distribution in the developing ovule before fertilization were investigated in Arabidopsis thaliana. Nucellar degeneration before fertilization occurs through vacuolar cell death and in an ordered degeneration fashion. This sequential nucellar degeneration is controlled by the signalling molecule auxin. Auxin efflux plays the core role in precisely controlling the spatiotemporal pattern of auxin distribution in the nucellus surrounding the FG. The auxin efflux carrier PIN1 transports maternal auxin into the nucellus while PIN3/PIN4/PIN7 further delivers auxin to degenerating nucellar cells and concurrently controls FG central vacuole expansion. Notably, auxin concentration and auxin efflux are controlled by the maternal tissues, acting as a key communication from maternal to filial tissue.

Plant Physiology, 2019

Stressful environments often lead to protein unfolding and the formation of cytotoxic aggregates ... more Stressful environments often lead to protein unfolding and the formation of cytotoxic aggregates that can compromise cell survival. The molecular chaperone heat shock protein (HSP) 101 is a protein disaggregase that cooperates with the small HSP (sHSP) and HSP70 chaperones to facilitate removal of such aggregates and is essential for surviving severe heat stress. To better define how HSP101 protects plants, we investigated the localization and targets of this chaperone in Arabidopsis (Arabidopsis thaliana). By following HSP101 tagged with GFP, we discovered that its intracellular distribution is highly dynamic and includes a robust, reversible sequestration into cytoplasmic foci that vary in number and size among cell types and are potentially enriched in aggregated proteins. Affinity isolation of HSP101 recovered multiple proteasome subunits, suggesting a functional interaction. Consistent with this, the GFP-tagged 26S proteasome regulatory particle non-ATPase (RPN) 1a transiently colocalized with HSP101 in cytoplasmic foci during recovery. In addition, analysis of aggregated (insoluble) proteins showed they are extensively ubiquitylated during heat stress, especially in plants deficient in HSP101 or class I sHSPs, implying that protein disaggregation is important for optimal proteasomal degradation. Many potential HSP101 clients, identified by mass spectrometry of insoluble proteins, overlapped with known stress granule constituents and sHSPinteracting proteins, confirming a role for HSP101 in stress granule function. Connections between HSP101, stress granules, proteasomes, and ubiquitylation imply that dynamic coordination between protein disaggregation and proteolysis is required to survive proteotoxic stress caused by protein aggregation at high temperatures.

HortScience, 1990

When plants experience high temperature stress, they respond by synthesizing a discrete set of pr... more When plants experience high temperature stress, they respond by synthesizing a discrete set of proteins called heat shock proteins (HSPs). This response is not unique to plants, but is observed in all other eukaryotes. It is now known that the HSPs are evolutionarily conserved proteins, and furthermore, that HSPs function not only during stress, but also during normal growth and development. My laboratory has characterized several of the major groups of HSPs in higher plants. We have cloned genes encoding plant HSP70 proteins and low molecular weight (LMW) HSPs (17-23 kDa). Using this information we have investigated the expression of HSPs both in the field, and under laboratory conditions which mimic field situations. We have determined the temperature limits for expression of HSPs in vegetative tissues, and have also found that HSPs are frequently produced in plant reproductive structures, even in the absence of stress. As a first step toward understanding HSP function, we have ch...

Phenotypic variation in stress response has been widely observed within species. This variation i... more Phenotypic variation in stress response has been widely observed within species. This variation is an adaptive response to local climates and is controlled by gene sequence variation and especially by variation in expression at the transcriptome level. Plants from contrasting climates are thus expected to have different patterns in gene expression. Acclimation, a pre-exposure to sub-lethal temperature before exposing to extreme high temperature, is an important adaptive mechanism of plant survival. We are interested to evaluate the gene expression difference to heat stress for plants from contrasting climates and the role of acclimation in altering their gene expression pattern. Natural Arabidopsis thaliana plants from low elevation mediterranean and high elevation montane climates were exposed to two heat treatments at the bolting stage: a) 45 oC: a direct exposure to 45oC heat; b) 38/45 oC: an exposure to 45oC heat after a 38oC acclimation treatment. Variation in overall gene expr...

Journal of Biological Chemistry, 1995

Current Biology, 1992

Antisense RNA was used to effectively inhibit ethylene production in tomato, and an inhibition of... more Antisense RNA was used to effectively inhibit ethylene production in tomato, and an inhibition of ripening was observed. This demonstrates that ethylene is the trigger of ripening and not a by-product.

Chemistry & Biology, 2010

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization o... more The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein.

Chemistry & Biology, 2008

The dynamics of protein complexes are crucial for their function yet are challenging to study. He... more The dynamics of protein complexes are crucial for their function yet are challenging to study. Here, we present a nanoelectrospray (nESI) mass spectrometry (MS) approach capable of simultaneously providing structural and dynamical information for protein complexes. We investigate the properties of two small heat shock proteins (sHSPs) and find that these proteins exist as dodecamers composed of dimeric building blocks. Moreover, we show that these proteins exchange dimers on the timescale of minutes, with the rate of exchange being strongly temperature dependent. Because these proteins are expressed in the same cellular compartment, we anticipate that this dynamical behavior is crucial to their function in vivo. Furthermore, we propose that the approach used here is applicable to a range of nonequilibrium systems and is capable of providing both structural and dynamical information necessary for functional genomics.

Molecular Biology and Evolution, 1999

A cDNA library was constructed with mRNA isolated from heat-stressed cell cultures of Funaria hyg... more A cDNA library was constructed with mRNA isolated from heat-stressed cell cultures of Funaria hygrometrica (Bryophyta, Musci, Funariaceae). cDNA clones encoding six cytosolic small heat shock proteins (sHSPs) were identified using differential screening. Phylogenetic analysis of these sHSP sequences with other known sHSPs identified them as members of the previously described higher plant cytosolic class I and II families. Four of the F. hygrometrica sHSPs are members of the cytosolic class I family, and the other two are members of the cytosolic class II family. The presence of members of the cytosolic I and II sHSP families in a bryophyte indicates that these gene families are ancient, and evolved at least 450 MYA. This result also indicates that the plant sHSP gene families duplicated much earlier than did the well-studied phytochrome gene family. Members of the cytosolic I and II sHSP families are developmentally regulated in seeds and flowers in higher plants. Our findings show that the two cytosolic sHSP families evolved before the appearance of these specialized structures. Previous analysis of angiosperm sHSPs had identified class-or family-specific amino acid consensus regions and determined that rate heterogeneity exists among the different sHSP families. The analysis of the F. hygrometrica sHSP sequences reveals patterns and rates of evolution distinct from those seen among angiosperm sHSPs. Some, but not all, of the amino acid consensus regions identified in seed plants are conserved in the F. hygrometrica sHSPs. Taken together, the results of this study illuminate the evolution of the sHSP gene families and illustrate the importance of including representatives of basal land plant lineages in plant molecular evolutionary studies.

The Plant Cell, 2005

We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaper... more We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 45°C after acclimation at 38°C, but also is sensitive to 38°C, which is a permissive temperature for wild-type and loss-of-function mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function mutations (Class 1) that eliminated the 38°C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2 suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors supports a functional link between the c...

Journal of Biological Chemistry, 2003

Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured protein... more Small heat shock proteins (sHsps) are ubiquitous molecular chaperones that bind denatured proteins in vitro, thereby facilitating their subsequent refolding by ATP-dependent chaperones. The mechanistic basis of this refolding process is poorly defined. We demonstrate that substrates complexed to sHsps from various sources are not released spontaneously. Dissociation and refolding of sHsp bound substrates relies on a disaggregation reaction mediated by the DnaK system, or, more efficiently, by ClpB/DnaK. While the DnaK system alone works for small, soluble sHsp/substrate complexes, ClpB/DnaK-mediated protein refolding is fastest for large, insoluble protein aggregates with incorporated sHsps. Such conditions reflect the situation in vivo, where sHsps are usually associated with insoluble proteins during heat stress. We therefore propose that sHsp function in cellular protein quality control is to promote rapid resolubilization of aggregated proteins, formed upon severe heat stress, by DnaK or ClpB/DnaK. * This work was supported by grants from the Deutsche Forschungsgemeinschaft (Leibnizprogramm and Bu617/15-1) and the Fond der Chemischen Industrie (to B. B. and A. M.

Journal of Biological …, 2004

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believ... more The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has ...

Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley... more Cytochemical and immunocytochemical methods were used to localize photosystems I and II in barley (Hordeum vulgare L. cv Himalaya) chloroplasts. PSI activity, monitored by diaminobenzidine oxidation, was associated with the lumen side of the thylakoids of both grana and stroma lamellae. The P700 chlorophyll a protein, the reaction center of PSI, was localized on thin sections of barley chloroplasts using monospecific antibodies to this protein and the peroxidase-antiperoxidase procedure. Results obtained by immunocytochemistry were similar to those of the diaminobenzidine oxidation: both grana and stroma lamellae contained immunocytochemically reactive material. Both the grana and stroma lamellae were also labeled when isolated thylakoids were reacted with the P700 chlorophyll a protein antiserum and then processed by the peroxidase-antiperoxidase procedure. PSII activity was localized cytochemically by monitoring the photoreduction of thiocarbamyl nitroblue tetrazolium, a reaction sensitive to the PSII inhibitor, DCMU. PSII reactions occurred primarily on the grana lamellae, with weaker reactions on the stroma lamellae. 'This research was supported in part by National Institutes of Health Grants GMO7 183 and GM23944 to R. S. A. Part of this work has been presented in abstract form (Vaughn, Vierling, Duke, Alberte 1982 Plant Physiol 69: S70). Mention ofa trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

Mitochondria play critical roles in eukaryotes in ATP generation through oxidative phosphorylatio... more Mitochondria play critical roles in eukaryotes in ATP generation through oxidative phosphorylation (OXPHOS) and also produce both damaging and signaling reactive oxygen species (ROS). Originating from endosymbiosis, mitochondria have their own reduced genomes that encode essential subunits of the OXPHOS machinery. MTERF (Mitochondrial Transcription tERmination Factor-related) proteins have been shown to be involved in organelle gene expression by interacting with organellar DNA or RNA in multicellular eukaryotes. We previously identified mutations in Arabidopsis thaliana MTERF18/SHOT1 that enable plants to better tolerate heat and oxidative stresses, presumably due to low ROS and reduced oxidative damage. To understand molecular mechanisms leading to shot1 phenotypes, we investigated mitochondrial defects of shot1 mutants and targets of the SHOT1 protein. shot1 mutants have problems accumulating OXPHOS complexes that contain mitochondria-encoded subunits, with complex I and complex ...

Cell Stress and Chaperones

Journal of Biological Chemistry

It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate. ... more It takes a dimer to tango: Oligomeric small heat shock proteins dissociate to capture substrate. Journal of Biological Chemistry jbc.RA118.005421. c the American Society for Biochemistry and Molecular Biology Use policy The full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-prot purposes provided that: • a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.