热激蛋白ClpB的结构和功能

ClpB是HSP100／Clp蛋白家族的一员,具有分子伴侣功能,通过溶解热胁迫下的蛋白聚集体,减少热激对细胞的伤害,ClpB与细胞的耐热性紧密相关。近年来,ClpB在植物中的功能成为研究的热点,但大多局限在植物胞质ClpB的研究,而对细胞器ClpB的研究较少。文章介绍了热激蛋白ClpB的结构、功能和植物热激蛋白ClpB的研究现状。     

植物热激蛋白90的结构和功能

文章从结构和功能两个方面介绍植物热激蛋白90的研究进展。     

结核杆菌小分子热休克蛋白Hsp16.3的高效自发再折叠和再组装

来自结核杆菌的小分子热休克蛋白Hsp16.3以九聚体的形式存在.用三种不同的强变性条件(100℃加热15 min,12 mol/L脲或8 mol/L盐酸胍处理4 h)将Hsp16.3变性, 然后通过冷却或透析使之复性,并利用孔径梯度聚丙烯酰胺凝胶电泳和圆二色性光谱比较了变性-复性前后Hsp16.3的各个层次高级结构.结果显示,变性的Hsp16.3几乎可以完全恢复至天然构象,这表明小分子热休克蛋白Hsp16.3具有很强的自发折叠和组装能力.     

Detection and Architecture of Small Heat Shock Protein Monomers

Background

Small Heat Shock Proteins (sHSPs) are chaperone-like proteins involved in the prevention of the irreversible aggregation of misfolded proteins. Although many studies have already been conducted on sHSPs, the molecular mechanisms and structural properties of these proteins remain unclear. Here, we propose a better understanding of the architecture, organization and properties of the sHSP family through structural and functional annotations. We focused on the Alpha Crystallin Domain (ACD), a sandwich fold that is the hallmark of the sHSP family.

Methodology/Principal Findings

We developed a new approach for detecting sHSPs and delineating ACDs based on an iterative Hidden Markov Model algorithm using a multiple alignment profile generated from structural data on ACD. Using this procedure on the UniProt databank, we found 4478 sequences identified as sHSPs, showing a very good coverage with the corresponding PROSITE and Pfam profiles. ACD was then delimited and structurally annotated. We showed that taxonomic-based groups of sHSPs (animals, plants, bacteria) have unique features regarding the length of their ACD and, more specifically, the length of a large loop within ACD. We detailed highly conserved residues and patterns specific to the whole family or to some groups of sHSPs. For 96% of studied sHSPs, we identified in the C-terminal region a conserved I/V/L-X-I/V/L motif that acts as an anchor in the oligomerization process. The fragment defined from the end of ACD to the end of this motif has a mean length of 14 residues and was named the C-terminal Anchoring Module (CAM).

Conclusions/Significance

This work annotates structural components of ACD and quantifies properties of several thousand sHSPs. It gives a more accurate overview of the architecture of sHSP monomers.     

热休克蛋白70的主要功能和应用前景

热休克蛋白70(HSP70)是生物体在各种应激条件下产生的蛋白之一,具有维持细胞自身稳定等多种生物学功能.随着研究的深入,在生物学的功能不断被发现的同时,HSP70的应用前景也变得越来越广泛.     

A Model for Small Heat Shock Protein Inhibition of Polyglutamine Aggregation

Polyglutamine (polyQ) repeat expansions that lead to the formation of amyloid aggregates are linked to several devastating neurodegenerative disorders. While molecular chaperones, including the small heat shock proteins (sHsp), play an important role in protection against protein misfolding, the aberrant protein folding that accompanies these polyQ diseases overwhelms the chaperone network. By generating a model structure to explain the observed suppression of spinocerebellar ataxia 3 (SCA3) by the sHsp αB-crystallin, we have identified key vulnerabilities that provide a possible mechanism to explain this heat shock response. A docking study involving a small bioactive peptide should also aid in the development of new drug targets for the prevention of polyQ-based aggregation.     

Late Onset of the Serological Response against the 18 kDa Small Heat Shock Protein of Mycobacterium ulcerans in Children

A previous survey for clinical cases of Buruli ulcer (BU) in the Mapé Basin of Cameroon suggested that, compared to older age groups, very young children may be less exposed to Mycobacterium ulcerans. Here we determined serum IgG titres against the 18 kDa small heat shock protein (shsp) of M. ulcerans in 875 individuals living in the BU endemic river basins of the Mapé in Cameroon and the Densu in Ghana. While none of the sera collected from children below the age of four contained significant amounts of 18 kDa shsp specific antibodies, the majority of sera had high IgG titres against the Plasmodium falciparum merozoite surface protein 1 (MSP-1). These data suggest that exposure to M. ulcerans increases at an age which coincides with the children moving further away from their homes and having more intense environmental contact, including exposure to water bodies at the periphery of their villages.     

人热休克蛋白70和热休克固有蛋白70的基因克隆和表达

目的:克隆人热休克蛋白70(HSP70)和热休克固有蛋白70(HSC70)基因,并在大肠杆茵中表达,获得重组蛋白.方法:用RT-PCR法从HepG2细胞中扩增HSP70及HSC70cDNA序列.测序后,将相应的cDNA插入pRSET-A表达载体,在大肠杆菌中表达,重组蛋白纯化后用SDS-PAGE及Western Blotting分析.结果:DNA序列结果显示.本研究所获得的HSP70及HSC70 cDNA序列与参考序列一致.将全长cDNA分别插入表达质粒后,转化BL21(DE3)细菌,在IPTG的诱导下,表达产物SDS-PAGE显示相应的分子量(70kDa)位置有明显的蛋白条带.Western Blotting结果证实了其为目的蛋白,经镍树脂柱纯化,获得了相应的重组多肽.结论:成功构建了原核表达重组质粒HSP70-pRSET-A和HSC70-pRSET-A,并获得了纯化的重组人HSP70和HSC70蛋白,为进一步研究这两种蛋白的结构、功能及临床应用奠定了基础.     

甜椒细胞质小分子量热激蛋白基因(CaHSP18)的cDNA克隆与表达

用RT-PCR和RACE-PCR技术,从热激处理的甜椒叶片总RNA中扩增出了细胞质小分子量热激蛋白(sHSP)全长779 bp的cDNA基因序列,包含一个480 bp开放阅读框,编码159个氨基酸.Southern杂交结果表明在甜椒基因组中有该基因的小的多基因家族.Northern结果显示该基因在甜椒根、茎、叶中的表达受热激和低温的诱导.原核表达分析表明该基因在高温以及低温条件下可以提高大肠杆菌的生存能力.     

Heat Shock Protein 70 Family: Multiple Sequence Comparisons, Function, and Evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus ``matches' 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins. Received: 29 January 1998 / Accepted: 14 May 1998     

Measurement of ATP generation and decay in Mycobacterium leprae in vitro

The intracellular ATP content of Mycobacterium leprae isolated from armadillo tissue was approximately 1.5 X 10(-16) g per bacillus. During in vitro incubation of bacilli at 4 degrees C, 33 degrees C or 37 degrees C there was an exponential decrease in ATP content, the rate depending on the medium and the temperature. M. leprae incorporated phosphate into ATP and into other nucleotide materials during in vitro incubation.     

Plantation Forestry under Global Warming: Hybrid Poplars with Improved Thermotolerance Provide New Insights on the in Vivo Function of Small Heat Shock Protein Chaperones

Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula × Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones.In spite of the vast importance of trees as a renewable source of biomaterials and energy, forestry is largely dominated by traditional approaches worldwide. These have been unable to keep up with demand over the last half century, leading to significant deforestation and global concern over carbon emissions (Bonan, 2008). According to the Food and Agriculture Organization of the United Nations (FAO), the net loss in forest area is now at some 5.2 million ha per year, an alarmingly high rate (Global Forest Resources Assessment, 2010 [www.fao.org/forestry/fra2010/]). Tree farming is gaining momentum to reverse this state of affairs, but current yields must be significantly improved for plantations to become a realistic and sustainable alternative to forest logging (Boerjan, 2005; Fenning et al., 2008; Strauss et al., 2009; Harfouche et al., 2011, 2012). Global warming makes this a challenging goal, as evidenced by the massive losses in crop production attributable to the increased temperatures of the last decades (Lobell et al., 2011). Climate-driven heat stress has also been recently identified as a major cause of forest die off (Anderegg et al., 2013). While climate trends make the establishment of high-yielding plantations more pressing than ever, improving the thermotolerance of valuable genotypes has proven difficult (Neale and Kremer, 2011; Harfouche et al., 2012). Apart from long juvenile periods and insufficient genomic resources, progress in this area has been hampered by our poor understanding of the biochemical mechanisms underlying thermotolerance. Forest biotechnology has great potential in this respect, as promising candidate genes can be safely assessed under controlled conditions. Tree breeding programs will integrate this knowledge in the foreseeable future to help improve elite genotypes and sidestep undesirable phenotypic variation (Fenning et al., 2008; Strauss et al., 2009; Harfouche et al., 2011, 2012). A successful example is the development of freeze-tolerant hybrid eucalyptus trees (Eucalyptus grandis × Eucalyptus urophylla) through a biotechnological approach (Hinchee et al., 2009).Extensive research has shown that high-temperature stress has a negative impact on nearly every aspect of plant growth, development, reproduction, and yield (for review, see Mittler et al., 2012). To protect cell function, plants have evolved a complex metabolic adjustment process known as the heat shock response (HSR; Kotak et al., 2007; Mittler at al., 2012). Alternatively, programmed cell death is activated in specific cells, resulting in leaf shedding or abortion of reproductive organs (Qu et al., 2009; Blanvillain et al., 2011). Whereas research in forest trees remains scarce, the ubiquitous and conserved HSR has been thoroughly studied in herbaceous plants (Larkindale and Vierling, 2008; Hu et al., 2009; Finka et al., 2011; Mittler et al., 2012). It is now well established that it involves multiple biochemical and regulatory pathways aimed at minimizing damage and preserving cellular homeostasis. Heat stress promotes protein unfolding and aggregation, and a major component of the HSR is the induction of molecular chaperones of the heat shock protein (HSP) family. HSPs share the ability to recognize and bind other proteins in nonnative states, thereby preventing or reversing aggregation as well as promoting efficient refolding pathways. Moreover, HSPs facilitate the degradation of proteins that cannot be properly folded by facilitating their delivery to cellular proteases.The most abundant and heterogeneous HSPs in plants are the widespread small heat shock proteins (sHSPs). At least 10 separate families have been described in both monocots and dicots, which include proteins localized to the cytoplasm (four classes), nucleus, chloroplasts, mitochondria, endoplasmic reticulum, and peroxisomes (for review, see Basha et al. [2012]; Waters [2013]). Along with their remarkable up-regulation under stressful conditions, the above features suggest important protective roles in virtually all cellular compartments. Yet, sHSPs remain one of the least well-understood families of molecular chaperones. With monomeric sizes of approximately 12 to 43 kD, plant sHSPs share a signature α-crystallin domain of approximately 90 amino acids and the ability to form large oligomers with a dynamic quaternary structure. The majority of structural and functional information derives from studies of cytosolic class I (CI) members, like wheat (Triticum aestivum) TaHSP16.9, for which the crystal structure of the native dodecamer has been solved (van Montfort et al., 2001). While researchers have demonstrated that most sHSPs can act as ATP-independent molecular chaperones, understanding their in vivo mode of action is complex (Basha et al., 2012; Waters, 2013). The prevailing view is that they prevent irreversible protein aggregation by interacting with unfolded proteins generated during stress. Recent evidence indicates that sHSP oligomers bind up to an equal weight of substrate proteins through a unique mechanism that involves substrate- and temperature-dependent remodeling of their own quaternary structure (Jaya et al., 2009; Stengel et al., 2010). Subsequent refolding of the substrates requires, at least in some cases, interaction with high-M_r ATP-dependent chaperones (Haslbeck et al., 2005; Basha et al., 2012). Thus, sHSPs would act primarily as holdases that keep nonnative proteins in a competent state until other downstream chaperones facilitate refolding. In vivo sHSP substrates remain to be identified in higher plants, although different lines of evidence suggest that they are structurally diverse (Basha et al., 2004; Haslbeck et al., 2004; Cheng et al., 2008; Jaya et al., 2009). Likewise, the role of sHSPs in mechanisms of thermotolerance has not yet been established. Reverse genetic approaches have led to inconsistent results in Arabidopsis (Arabidopsis thaliana) and other herbaceous species, ranging from variable protection (Malik et al., 1999; Sanmiya et al., 2004; Zhao et al., 2007; Jiang et al., 2009) to no detectable effects at all (Härndahl et al., 1999; Sun et al., 2001).CsHSP17.5, a major CI sHSP in chestnut trees (Castanea sativa), exhibits features that make it a good candidate to improve heat stress adaptation. First, its accumulation in adult trees increases substantially during spring and early summer, reaching a peak at the hottest time of the year (field conditions). Second, the corresponding gene is strongly activated by heat stress (growth chambers). Third, it significantly improves Escherichia coli viability under heat stress conditions. Fourth, it accumulates at unusually high levels in both stems and seeds, where it reaches levels comparable to those of major storage proteins (Collada et al., 1997; Soto et al., 1999; Lopez-Matas et al., 2004). Besides suggesting a relevant role in thermotolerance, these features indicate that CsHSP17.5 can accumulate in vivo for long periods of time. In vitro experiments have shown, additionally, that the native protein can stand repeated cycles of stressful conditions without losing chaperone activity (Lopez-Matas et al., 2004). The fact that CsHSP17.5 has never been identified as an allergen, despite accumulating so abundantly in mature chestnuts (according to FAO, humans consume over half a million metric tons a year), adds to its biotechnological potential. Here, we show that constitutive overexpression of native CsHSP17.5 substantially enhances the basal thermotolerance of hybrid poplar (Populus tremula × Populus alba) without causing negative pleiotropic effects that might affect plantation yields. A significant improvement of plant performance in vitro and ex vitro was also observed, even in the absence of thermal stress. Our results shed new light on the in vivo roles of sHSPs. In addition, they represent, to our knowledge, the first example of biotechnological manipulation of thermotolerance in forest trees.     

结核杆菌Hsp65与人IL-2融合蛋白在耻垢杆菌表达

目的：构建能够分泌表达结核分枝杆菌热休克蛋白65（Hsp65）与人IL-2融合蛋白的重组耻垢分枝杆菌（recombinant Mycobacterium Smegmatis,rMs）。方法：用EcoRV和HindIII双酶切含Hsp65．IL-2融合基因的pPRO-hsp65-IL-2载体,回收目的基因片断Hsp65-IL-2,并将其亚克隆入同样双酶切的大肠埃希菌-分枝杆菌穿梭分泌表达载体pDE22中。重组质粒pDE22-hsp65-IL-2酶切鉴定正确后,电穿孔转化MS感受态,潮霉素抗性压力筛选阳性rMs。Westem—blot鉴定rMs培养上清蛋白中目的蛋白的表达。结果：重组pDE22-hsp65-IL-2质粒酶切后可获得约2000bp片段,与预期大小一致。Western-blot结果表明,rMs培养上清蛋白中有特异性反应条带,大小为78kD,与Hsp65-IL-2融合蛋白大小相一致。结论：成功构建了大肠埃希菌．分枝杆菌穿梭分泌表达载体pDE22-hsp65-IL-2,为该rMs的免疫学特性及抗结核分枝杆菌感染的保护效果研究奠定了基础。     

The Function of Ile-X-Ile Motif in the Oligomerization and Chaperone-Like Activity of Small Heat Shock Protein AgsA at Room Temperature

Small heat shock proteins assemble as large oligomers in vitro and exhibit ATP-independent chaperone activities. Ile-X-Ile motif is essential in both the function and oligomer formation. AgsA of Salmonella enterica serovar Typhimurium has been demonstrated to adopt large oligomeric structure and possess strong chaperone activity. Size exclusion chromatography, non-denaturing pore gradient PAGE, and negatively stain electron microscopic analysis of the various C-terminal truncated mutants were performed to investigate the role of Ile-X-Ile motif in the oligomer assembly of AgsA. By measuring the ability to prevent insulin from aggregating induced by TCEP, the chaperone-like activity of AgsA and the C-terminal truncated mutants at room temperature were determined. We found that the truncated mutants with Ile-X-Ile motif partially or fully deleted lost the ability to form large oligomers. Contrast to wild type AgsA which displayed weak chaperone-like activity, those mutants shown significantly enhanced activities at room temperature. In summary, biochemical experiment, activity assay and electron microscopic analysis suggested that Ile-X-Ile motif is essential in oligomer assembly of AgsA and might take the role of an inhibitor for its chaperone-like activity at room temperature.     

胞壁表达HSP65-IL-2融合蛋白的重组耻垢分枝杆菌的构建和鉴定

目的：获得胞壁表达结核分枝杆菌热激蛋白65（HSP65）和人白细胞介素2（IL-2）融合蛋白的重组耻垢分枝杆菌。方法：将HSP65和IL-2融合基因克隆入大肠杆菌-卡介苗（E．coli-BCG）穿梭质粒pCW,构建成重组质粒HSP65-IL-2-pCW,电穿入耻垢分枝杆菌,经潮霉素抗性筛选和PCR方法选取阳性克隆;用间接免疫荧光法对重组耻垢分枝杆菌进行表型鉴定;用重组耻垢分枝杆菌免疫BALB／c小鼠,检测其诱导的抗体水平。结果：筛选获得的重组耻垢分枝杆菌增殖特性与普通耻垢分枝杆菌无明显区别;与抗人的IL-2抗体和HSP65抗体均可形成免疫印迹条带;间接荧光染色后,可见细菌表面有极强的绿色荧光;重组耻垢分枝杆菌免疫BALB／c小鼠2周后,小鼠血清中HSP65抗体平均滴度为1：4000,而生理盐水组的抗体几乎为阴性。结论：结核分枝杆菌HSP65和人IL-2融合基因在耻垢分枝杆菌胞壁获得表达,有望为结核病的预防提供有效的疫苗。     

The Small Heat Shock Protein Hsp27 Affects Assembly Dynamics and Structure of Keratin Intermediate Filament Networks

The mechanical properties of living cells are essential for many processes. They are defined by the cytoskeleton, a composite network of protein fibers. Thus, the precise control of its architecture is of paramount importance. Our knowledge about the molecular and physical mechanisms defining the network structure remains scarce, especially for the intermediate filament cytoskeleton. Here, we investigate the effect of small heat shock proteins on the keratin 8/18 intermediate filament cytoskeleton using a well-controlled model system of reconstituted keratin networks. We demonstrate that Hsp27 severely alters the structure of such networks by changing their assembly dynamics. Furthermore, the C-terminal tail domain of keratin 8 is shown to be essential for this effect. Combining results from fluorescence and electron microscopy with data from analytical ultracentrifugation reveals the crucial role of kinetic trapping in keratin network formation.     

BOBBER1 Is a Noncanonical Arabidopsis Small Heat Shock Protein Required for Both Development and Thermotolerance

Plants have evolved a range of cellular responses to maintain developmental homeostasis and to survive over a range of temperatures. Here, we describe the in vivo and in vitro functions of BOBBER1 (BOB1), a NudC domain containing Arabidopsis (Arabidopsis thaliana) small heat shock protein. BOB1 is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. Because BOB1 loss-of-function mutants are embryo lethal, we used a partial loss-of-function allele (bob1-3) to demonstrate that BOB1 is required for organismal thermotolerance and postembryonic development. Recombinant BOB1 protein functions as a molecular chaperone and prevents the aggregation of a model protein substrate in vitro. In plants, BOB1 is cytoplasmic at basal temperatures, but forms heat shock granules containing canonical small heat shock proteins at high temperatures. In addition to thermotolerance defects, bob1-3 exhibits pleiotropic development defects during all phases of development. bob1-3 phenotypes include decreased rates of shoot and root growth as well as patterning defects in leaves, flowers, and inflorescence meristems. Most eukaryotic chaperones play important roles in protein folding either during protein synthesis or during cellular responses to denaturing stress. Our results provide, to our knowledge, the first evidence of a plant small heat shock protein that has both developmental and thermotolerance functions and may play a role in both of these folding networks.Plants are autotrophic sessile organisms that depend on sunlight for their energetic needs. One consequence of this lifestyle is that plants are often subjected to high temperature stress, especially in dry conditions when transpirational cooling is limited. At a cellular level, elevated temperatures result in changes in protein structure that can result in the exposure of normally buried hydrophobic residues. As a consequence of thermal denaturation, proteins may aggregate and cease to function normally. A universal response to temperature-induced protein unfolding in all living organisms is the production of heat shock proteins (HSPs). HSPs are molecular chaperones that provide organismal thermotolerance by preventing the denaturation and aggregation of target proteins as well as facilitating protein refolding. Highly conserved HSPs are found in all organisms and include the small HSP (sHSP) as well as the Hsp60, Hsp70, Hsp90, and Hsp100 families (Baniwal et al., 2004; Taiz and Zeiger, 2006). Members of the sHSP family are defined by their small size (12–43 kD), their ability to prevent protein aggregation, and by a conserved α-crystallin domain (ACD). Plants are unusual in the large number of ACD-containing sHSPs encoded by their genomes: Arabidopsis (Arabidopsis thaliana) has 19 compared to 10 in humans, four in Drosophila melanogaster, and one or two in bacteria (Haslbeck et al., 2005).Although the biochemical activity of plant sHSPs has been well characterized (Lee et al., 1995, 1997; Basha et al., 2004; Siddique et al., 2008), little is known about the in vivo functions of plant sHSPs, perhaps due to functional redundancies in this large gene family. Apart from temperature-dependent changes in hypocotyl elongation, which reflects the ability of cells to expand, no developmental roles for a sHSP have been reported in plants (Jenks and Hasegawa, 2005; Dafny-Yelin et al., 2008). In addition to redundancy, a lack of known developmental functions for plant sHSPs may also be a result of the fact that most are only expressed in response to heat or other stresses. Exceptions include a subset of sHSPs expressed during seed and pollen maturation, developmental stages that involve desiccation (Wehmeyer and Vierling, 2000; Dafny-Yelin et al., 2008). However, since most plant sHSPs are not expressed under nonstress conditions, they are unlikely to affect normal growth and development (Swindell et al., 2007).BOBBER1 (BOB1; At5g53400) is an essential gene required for the normal partitioning and patterning of the apical domain of the Arabidopsis embryo. In bob1-1 and bob1-2 null mutants, meristematic identity is expanded into the portion of the embryo that would normally form the seedling leaves (cotyledons), which in turn are never established. Auxin gradients are never established in bob1 mutant embryos. However, since there are multiple feedback loops involved in auxin signaling and transport, it is unclear whether the lack of auxin maxima in bob1 mutants is a direct or indirect result of a lack of BOB1 activity (Jurkuta et al., 2009). BOB1 encodes a protein with C-terminal homology to NudC, a protein identified in a screen for genes required for nuclear migration in Aspergillus nidulans. Genes with homology to NudC have been shown to interact with dynein microtubule motors. In mammalian tissue culture systems, interference with NudC-like gene function results in defects in chromosome segregation and cytokinesis (Aumais et al., 2003; Nishino et al., 2006; Zhou et al., 2006). The NudC domain has predicted structural homology with the α-crystallin/p23 protein families (Garcia-Ranea et al., 2002), which includes the ACD-containing sHSPs. The ACD, originally identified in the α-crystallin chaperone of the vertebrate eye lens, forms a structure consisting of two antiparallel β-sheets in a sandwich (Scharf et al., 2001; Haslbeck et al., 2005). The NMR structure of the mouse NudC homolog (PDB 1wfi) has the same β-sheet sandwich structure that provides support for the predicted structural homology between NudC domains and ACDs. These observations suggest that NudC domain proteins might share conserved functions with sHSPs. Support for this hypothesis comes from Caenorhabditis elegans where the NudC homolog NUD-1, an essential gene, displays protein chaperone activity in vitro (Faircloth et al., 2009).Here, we use bob1-3, a partial loss-of-function allele, to show that BOB1 is required for normal development and meristem function after embryogenesis. To determine whether BOB1 functions as a protein chaperone, we characterized the in vitro activity of BOB1 protein. We also investigated the thermotolerance functions of BOB1 using bob1-3 and used a BOB1:GFP line that is biologically active to document that BOB1 protein is incorporated into heat shock granules (HSGs) at high temperatures. All of these data suggest that BOB1 encodes a novel sHSP with dual functions in development and thermotolerance. To our knowledge, this is the first demonstration of a developmental patterning function for a plant sHSP.