- Email: [email protected]
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protein. As yet, there is no direct evidence that p21 functions by a cycle of GTP binding and hydrolysis, and we do not know what the relative biological effects of p21.GTP vs p21.GDP are. Nevertheless, the biochemical analysis of normal and oncogenic ras proteins has provided insight on ras function and the importance of p21 interaction with GTP. These findings indicate a direction and biochemical framework to explore the mechanism by which ras transforms mammalian cells.
7 Hughes, S. M. (1983) FEBS Lett. 164, 1--8 8 Gilman, A. G. (1984) Cell 36, 577-579 9 Stryer, L. (1983) Cold Spring Harbor Syrup. Quant. Biol. 48, 841-852 10 Dhar, R., Nieto, A., Koller, R., DeFeo-Jones, D. and Scolnick, E. M. (1984) Nucleic Acids Res 12, 3611-3618 11 Willumsen, B. M., Norris, K., Papageorge, A.G., Hubbert, N.L. and Lowy, D.R. (1984) EMBO J. 3, 2581-2585 12 Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D. and Gilman, A. G. (1984) Science 226, 860-862 13 Halliday, K. R. (1984) J. Cyclic Nucleotide and Protein Phosphorylation Res. 9, 435--448 14 Seeburg, P. H., Colby, W. W., Capon, D. J., Goeddel, D. V. and Levinson, A. D. (1984) References Nature 312, 71-75 1 Cooper, G.M. and Lane, M.A. (1983) 15 Fasano, O., Aldrich, T., Tamanoi, F., Biochim. Biophys. Acta 738, 9-20 Taparowsky, E., Furth, M. and Wiglet, M. 2 Shih, T. Y. and Weeks, M. O. (1984) Cancer (1984) Proc. Natl Acad. Sci. USA 81, Invest. 2, 109-123 4008--4012 3 DeFeo-Jones, D., Scolnick, E. M., Koller, R. 16 Temeles, G.L., Gibbs, J.B., D'Alonzo, and Dhar, R. (1983) Nature 306. 707-709 J. S., Sigal, I. S. and Scolnick, E. M. (1985) 4 Powers, S., Kataoka, T., Fasano, O., Nature 313,700-703 Goldfarb, M., Strathern, J., Broach, J. and 17 Papageorge, A., Lowy, D. and Scolnick, Wigler, M. (1984) Cell 36, 607-612 E. M. (1982)Z Virol. 44, 509-519 5 Neuman-Silberberg, F.S., Schejter, E., Hoffmann, F. M. and Shilo, B-Z. (1984) Cell 18 Finkel, T., Der, C.J. and Cooper, G. M. (1984) Cell 37, 151-158 37, 1027-1033 6 Reymond, C.D., Gomer, R.H., Mehdy, 19 Gibbs, J.B., Sigal, I.S., Poe, M. and Scolnick, E. M. (1984) Proc. Natl Acad. Sci. M.C. and Firtel, R.A. (1984) Cell 39, USA 81, 5704-5708 141-148
20 McGrath, J.P., Capon, D.J., Goeddel, D. V. and Levinson,A. D. (1984)Nature 310, 644-649 21 Mantle, V., Yamazaki, S. and Kung, H-F. (1984) Proc. Natl Acad. Sci. USA 81, 6953-6957 22 Sweet, R. W., Yokoyama, S., Kamata, T., Feramisco, J. R., Rosenberg, M. and Gross, M. (1984) Nature 311,273-275 23 Stacey, D. W. and Kung, H-F. (1984) Nature 310, 508-511 24 Feramisco, J.R., Gross, M., Kamata, T., Rosenberg, M. and Sweet, R. W. (1984) Cell 38, 109-117 25 Poe, M., Scolnick, E. M. and Stein, R. B. (1985) J. Biol. Chem. 260, 3906-3909 26 Gibbs, J. B., Ellis, R. W. and Scolnick,E. M. (1984) Proc. Natl Acad. Sci. USA 81, 2674-2678 27 Temeles, G. L., DeFeo-Jones, D., Tatchell, K., Ellinger, M. S. and Scolnick, E. M. (1984) Mol. Cell. Biol. 4, 2298-2305 28 Tamanoi, F., Walsh, M., Kataoka, T. and Wigler, M. (1984) Proc. Nag Acad. Sci. USA 81, 6924-6928 29 Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J. and Wigler, M. (1985) Cell 40, 19-26 30 DeFeo-Jones, D., Tatchell, K., Robinson, L. C., Sigal, I. S., Vass, W. C., Lowy, D. R. and Scolnick, E, M. (1985) Science 228, 179-184
Heat shock in plants
exception of the genes for the 83-85 kDa class of HS proteins, none of these genes contains introns. (3) When tissues are returned to their normal growing temperature, synthesis of HS proteins ceases, synthesis of other proteins recovers gradually, and the HS m R N A s decay fairly rapidly with a halflife of less than 2 hours. (4) Heat treatments which induce HS protein synthesis also lead to the development of thermal tolerance - the ability of an organism to withstand a normally lethal temperature if it is first given a HS at a non-lethal temperature.
Janice A. Kimpel and Joe L. Key The heat shock response is a ubiquitous phenomenon in all prokaryotes and eukaryotes that have been studied. In higher plants, the general characteristics o f this response have much in c o m m o n with those o f other organisms, including a probable role in the development o f short-term thermal tolerance. A s research on the response in higher plants continues, the focus is on understanding the regulation o f the heat shock genes and the function o f the heat shock proteins.
Over the past two decades, the heat shock (HS) response has been intensively studied, first in Drosophila and subsequently in a wide diversity of animals and microorganisms (for reviews, see Refs 1-3). In the last five years, the characteristics of the HS response in plants have been investigated in many species, including maize, tomato, cotton, tobacco, carrot, Chlamydomonas, soybean, pea and millet (see Ref. 4 [and Refs therein] and Ref. 5). Thus, it is now possible to conduct a comparative analysis of this response between plants and other organisms. The HS response of plants shares J. A. Kimpel and J. L. Key are at the Department of Botany, University of Georgia, Athens, GA 30602, USA. J.A. Kimpel is currently at the Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
many parameters with the responses to HS that have been described for Drosophila, animals and yeast: (1) Immediately following an abrupt shift of 8-10°C above the normal growing temperature, synthesis of a new group of proteins, the heat shock proteins (HS proteins) is induced and, at the same time, there is a decline in synthesis of the normal complement of cellular proteins. (2) The synthesis of these HS proteins is due to the transcription of a new set of genes, the HS genes. Many of these genes have now been sequenced, and a consensus promoter or enhancer-like sequence has been identified in the 5' flanking regions (for review, see Ref. 6). Evidence is accumulating that this core 15 bp sequence is critical to the heatinducibility of these genes. With the
Thus, the general phenotype of the HS response is highly conserved in all organisms that have been studied. This suggests that there is a fundamental role for HS proteins in cenular function during high temperature stress and possibly during recovery from the stress. However, the definition of such a function for these proteins has so far eluded researchers. We present here a concise review of recent results in research on the HS response in plants that might lead to the development of testable hypotheses about the function of these proteins and the regulation of the response.
The heat shock proteins of plants All organisms produce HS proteins that can roughly be divided into two
~) 1985.ElsevierSciencePublishersB.V. Amsterdam IB76- 5067/85/$02.00
TIBS - September 1985
354 groups: the high-molecular-weight (HMW) and the low-molecular-weight (LMW) HS proteins. In plants, the abundant HS proteins are a complex group of LMW proteins which resolve into more than 30 polypeptides during two-dimensional O'Farrell gel electrophoresis. The majority of these LMW proteins range in size from 15 to 18 kDa, but there are also HS proteins of 21, 24 and 27 kDa. The exact profile and complexity of LMW HS proteins varies among plant species, but a relatively high amount of this group of proteins is observed in all plant species4. All tissues of the plant seem to produce the full spectrum of HS proteins with the noted exceptions of pollen from Tradescant/a and maize, which do not produce any HS proteins (pp. 321-327 in Ref. 3 and Ref. 7). If this proves to be a general rule for plant pollen, it may have considerable physiological significance, particularly under field conditions. The abundance of these LMW proteins contrasts with the situation in mammalian, insect and yeast cells. In these organisms, the abundant HS proteins are HMW proteins of 65, 68, 70, 82-84, 90 and 100-110 kDa, The 70 kDa protein is by far the most abundant of all the HS proteins in these organisms, and antibodies to the protein isolated from chick cells cross-react with the 70 kDa HS proteins from Dictyostelium, Drosophila, human cell lines, and higher plants2. The HMW proteins of plants seem less complex than the LMW proteins. For a given plant species, the HMW proteins resolve into less than ten polypeptides during two-dimensional electrophoresis. There is at least one polypeptide of 90-110 kDa, one or two polypeptides of 80-90 kDa, and usually two or three polypeptides of 68-75 kDa. Heat shock proteins are synthesized very soon after organisms are placed at the HS temperature. In etiolated soybean seedlings, we have detected the synthesis of HS proteins within the first 15 min at 40°C. If these seedlings are retumed to their normal growing temperature of 28°C after between 30 rain and 2 h at 40°C, synthesis of the HS proteins declines dramatically. After 3-4 h at 28°C, synthesis of HS proteins is no longer detectable. However, the HS proteins that were made during this time are quite stable, and the majority are present in the tissue for at least 20 h8. In plants, the actual temperature at which HS proteins are maximally induced varies with each species, but it is correlated with the normal growing temperature. Pea, a cool-season species, maximally induces HS proteins around
37°(2, while millet, a warm-season grass, maximally induces HS proteins around 45°C. For maize and soybean, the optimal temperature is 40°C4. In general, synthesis of HS proteins is first detectable at temperatures about 5°C above the normal growing temperature. Synthesis of HS proteins as a percentage of total protein synthesis increases strongly as the temperature of the HS approaches the optimal temperature. Above this optimum, total protein synthesis drops off precipitously, but the majority of proteins synthesized at these higher temperatures are HS proteins. The synthesis of HS proteins results from the production of new mRNAs in the cell at the higher temperatures. In soybean seedlings, the HS mRNAs for the LMW proteins become very abundant during a 2-h HS, and this facilitated the isolation of HS cDNA clones by differential screening9. Using these cDNAs as probes, HS mRNAs can be detected within the first 5 min of a HS at 40°C. The steady state concentration of these LMW HS mRNAs increases steadily during 3-4 h of HS, then levels off and gradually declines with continued heat treatment. We do not know whether this reflects a stabilization of the HS mRNAs or a balance between synthesis and degradation of the mRNAs. In Drosophila, HS mRNAs were stable at 37°C, but they rapidly destabilized to a half-life of about 2 h when cells were returned to 25°C (Ref. 10). We have not been able to measure the half-life of the soybean HS mRNAs directly, but when seedlings are returned to 28°C, HS mRNA levels decline with an apparent half-life of 1-2 h (Ref. 8). By definition, HS proteins are a new set of proteins rapidly and abundantly produced in response to a HS, but in many organisms other stresses, such as ethanol, anoxia, arsenite, or heavy metal ions, will also induce the synthesis of HS proteins. However, with one exception, we have not detected synthesis of HS proteins in soybean seedlings under other stress conditions using incorporation of radioactive amino acids into proteins in vivo. It is possible to detect the HS mRNAs during these different stresses, but the amount of HS mRNAs is much less than that found during HS 11. Arsenite is the exception to these results; soybean seedlings incubated for 3 h in 50 ~M arsenite synthesize nearly the full spectrum of HMW and LMW HS proteins. We do not yet understand why arsenite in particular induces the HS proteins in soybean while many other stresses, which are effective in many other organisms,
do not appear to do so. Heat shock proteins and thermal tolerance From a physiological point of view, the only biological function assigned to HS proteins to date is their possible role in mediating the expression of thermal tolerance. In soybean seedlings, an absolute correlation has been found between the synthesis and accumulation of HS proteins and the ability to survive short heat treatments at otherwise lethal temperatures. It has long been known that a short period of high temperature (e.g. several minutes at 45°C, or 30 s at 50°C12), usually followed by some time at the normal growing temperature, could protect the plant from a subsequent heat treatment at an otherwise lethal temperature. We have recently demonstrated that a short HS at 45°C for 2-10 min triggers the synthesis of HS proteins. Interestingly, in these experiments, HS protein synthesis does not decline dramatically when the plants are returned to 28°C, as it does after a HS at 40°C. Instead, synthesis of HS proteins continues at high levels for approximately 2-3 h at 28°C, and then it declines gradually over the next 2-4 h. Our working hypothesis is that once the HS response is triggered, the accumulation of HS proteins is quantitatively monitored by the plant cells. When sufficient HS proteins are present in the cell to fulfil their protective function, HS protein synthesis stops. Such a hypothesis is supported by our additional observations that during prolonged heat treatment at 40°C, the synthesis of HS proteins declines after 3--4 h and is not detected after about 6 h. It is also supported by the results mentioned earlier regarding the quantitative, temperaturedependent aspects of the HS response. In some way, the organism must be able to measure its own temperature (either directly or as a differential) and mediate the extent of the HS response appropriately. It is possible that another product accumulates with the HS proteins during heat treatment which is responsible for the observed thermal tolerance. One test of this alternative hypothesis is to induce the synthesis of HS proteins without a heat treatment. This has been done in yeast using ethanol, in CHO cells using ethanol, arsenite or hypoxia (see Fig. 19 in Ref. 1 and references therein), and in soybean seedlings using arsenite '3. In all cases, thermal tolerance was observed after a pretreatment with the other stress agent. However, it is still possible to argue that factors other than
TIBS - September 1985 HS proteins are the critical determinants; perhaps stronger evidence comes from studies on mutants lacking the HS response. In Dictyostelium, a mutant was isolated which did not develop thermal tolerance to lethal temperatures when given a HS at a non-lethal temperature 14. This mutant did not synthesize the 26-32 kDa LMW HS proteins at all, and synthesis of the HMW proteins was only mildly enhanced during HS, compared to the wild-type. In E. coli, synthesis of the HS proteins is positively regulated by the htpR (hin) gene product. Mutants of E. coli lacking this gene product also fail to acquire thermal resistance during HS at a permissive temperature ~5. A double mutant of Saccharomyces cerevisiae was constructed by insertion mutagenesis at the two major 70 kDa HS protein genes~6. No synthesis of 70 kDa proteins was detectable in the mutant, yet it was as resistant as the wild-type to a brief exposure at a very high temperature after a preincubation at an intermediate temperature. Protein synthesis was still required for the acquisition of this resistance, and it was suggested that other HS proteins may be essential for protection against short-term exposure to extremely high temperatures. It is quite possible that different HS proteins have separate functions during heat treatments at various temperatures. Some HS proteins are also synthesized at specific times during development (e.g. in Drosophila) in the absence of any heat stress. It is feasible that some or all of the proteins identified as HS proteins may have additional roles in cellular function which might make selection of mutants more difficult. Another approach to understanding the role of HS proteins in thermal protection is to look at plants in the field. Plants are obligately and daily subjected to fluctuations in temperature that can be as much as 20-25°C. We have recently shown that soybean plants in the field synthesize HS mRNAs and proteins when air temperatures approach 40°C (Kimpel and Key, unpublished results). Plants in irrigated fields, which can avoid possibly deleterious effects of high air temperature by lowering their leaf temperature through evapotranspiration, have lower concentrations of HS mRNAs than nonirrigated plants. Thus, plants in the field and in the laboratory respond to transient increases in temperature by inducing the synthesis of HS proteins, and the extent of the response is determined by the severity of the heat treatment. Natural plant populations display a
355 wide range of heat tolerance, and much elegant research has focused on the physiological basis of the ability of some species to survive hot, often desert-like environments (for review, see Ref. 17). Is such heat adaptation related to HS proteins? There is as yet no research on the production of HS proteins in strongly heat tolerant species, but work on these plants indicates that other mechanisms are critical for adaptation. We hypothesize that the production of HS proteins is a short-term, induced response to heat stress at temperatures above the range of normal growing tem-~ peratures. We would predict that plants, normally adapted to grow in a very warm environment would also synthesize HS proteins at temperatures 8--10°C above the median ambient temperature. Because of the very high abundance and complexity of the LMW HS proteins of plants, it was originally suggested that these proteins might be unique to plants. The amino acid compositions of three different soybean HS proteins of about 18 kDa are over 90% homologous, but when they are compared to the 22 and 23 kDa proteins of Drosophila, the overall homology is less than 40%. However, hydropathy plots indicate four major regions of strong similarity. In the most hydrophobic domain, there is a striking conservation of sequence: Asp--Gly-Val-Leu-Thr(in the soybean genes, an Asn replaces Asp; Nagao, Czarnecka, Gudey, Schoffl and Key, unpublished results). This same sequence is found in a hydrophobic domain of the bovine a-crystallin proteinTM. This may be a clue to the function of HS proteins, since et-crystallins, major components of the vertebrate eye lens, are also LMW proteins that form large aggregates in the cells of the lens. Perhaps, LMW HS proteins also form large aggregates that perform a structural role in stabilizing various cellular constituents. In plants and Drosophila, there is evidence for such aggregation of HS proteins. In Drosophila, a 'nonnuclear structure' containing many HS polypeptides assembles and copurifies with nuclei during HS (see pp. 235-242 in Ref. 3). In tomato cell cultures and leaves, the LMW HS proteins form aggregates in the cytoplasm known as HS granules. These are only visible in the cells under HS conditions when HS proteins are presentL We have recently confirmed the existence of such large aggregates of the LMW HS proteins in soybean seedlings under HS conditions. Thus, at least one function of the LMW HS proteins could be structural. These
large aggregates in/he cell may serve as transient cellular matrices in which various cellular organelles and compartments are held and thereby stabilized. Such matrices would be maintained only during HS; once the cell temperature dropped back to normal, these aggregates would dissociate, allowing rapid recovery of normal cellular function. It is also clear that individual HS proteins become associated with specific organelles during HS. The 70 kDa protein of Drosophila concentrates in the nucleus during HS. Upon recovery from HS, this protein leaves the nucleus, but it rapidly reassociates with the nucleus during a second HS 19. In soybean seedlings, the 70 kDa HS proteins and the group of 15--18 kDa HS proteins become associated with nuclei only during HS. The 15-18 kDa HS proteins and the 21 and 24 kDa HS proteins become associated with the mitochondria during HS, but while the 15-18 kDa HS proteins dissociate from the mitochondrial fraction during recovery from HS, the 21 and 24 kDa proteins remain associated with the mitochondria after the seedlings are returned to the normal growing temperature ~3. We have recently identified another class of nuclear-encoded HS proteins which are specifically transported into chloroplasts 20. Maize, pea and soybean have major HS polypeptides in the chloroplasts that are between 23 and 25 kDa; soybean has an additional polypeptide of 27 kDa. Thus, some HS proteins associate with mitochondria during HS but do not dissociate upon recovery and some HS proteins are taken up by chloroplasts in an in vitro system. These results strongly suggest that these organelle-specific HS proteins are synthesized as precursors and processed during transport, leaving them irreversibly localized within the organeUe. In Drosophila, the localization of HS proteins is not strictly temperaturedependent, since anoxia also leads to concentration of the 70 kDa HS protein in the nucleus. However, in soybean seedlings and Drosophila 21, HS proteins synthesized during arsenite treatment are not localized to any organelle. When arsenite-treated soybean seedlings are subsequently heat-shocked, HS proteins synthesized during arsenite treatment rapidly localize with organelles, including the nuclei and mitochondria. While it seems likely that the function of HS proteins in thermal tolerance requires localization, it is not clear that such localization is a component of other physiological stresses.
356
Regulation of the HS response While many parameters of the HS response of plants have been investigated, there is little information on the mechanisms of regulation of the response. Regulation dearly occurs at both transcription and translation. Results from other organisms and our own research on soybeans indicate that during HS, normal cellular mRNAs persist in the cells but are translated very inefficiently if at all. In soybean seedlings, the level of actin mRNAs remains unchanged during HS, but the mRNA levels for the small subunit of ribulose bisphosphate carboxylase and an auxinregulated gene decrease dramatically during HS. The total poly(A)RNA content remains fairly constant during HS, and since HS mRNAs represent over 20% of the total poly(A)RNA pool after a 2-h HS, the concentrations of many normal poly(A)RNAs must decline. Thus, there must be a selective mechanism for preferential initiation and/or elongation of HS proteins on HS mRNA, relative to the population of normal mRNAs. In Drosophila, ceilfree lysates from heat-shocked cells do selectively translate HS mRNAs from a pool of total cellular mRNAs 22. When tissues are returned to normal temperatures after HS, the normal cellular mRNAs are once again efficiently translated. In soybean seedlings under prolonged heat treatment, HS mRNAs cease to be translated after about 6 h, and the translation of normal mRNAs recovers. The molecular basis for such translational regulation is not known. There has been little success in developing translation systems in vitro for more than a few plant species, so the existence of discriminating translational mechanisms has not been investigated in plants. The absence of HS mRNAs in tissues that have not been heat-shocked indicates that the induction of the HS response is obligately regulated at the level of transcription. Recently, factors have been identified that interact with RNA polymerase II and/or specific sequences in the regulatory regions of the HS genes to mediate the transcription of these genes during HS 2a.24. The identification of consensus, regulatory sequences has been the focus of much recent research (for review see Ref. 6), and we have recently completed analyses of several LMW HS genes of soybean (Ref. 25, and Nagao, R. T. et al., unpublished results). The 5' flanking regions of the soybean genes contain a sequence highly homologous to the HS consensus sequence identified in Drosophila, and it is located in a comparable
T I B S - September 1985
position to the T A T A box in the Drosophila hsp70 gene. There are additional regions of high homology to the HS consensus sequence located further upstream in the soybean LMW HS genes, and these may be important in regulating the level of expression of these genes. Recently, additional regulatory regions upstream of the HS consensus sequence have been identified that are needed for efficient promoter utilization2627. There are also numerous sequences in the 5' flanking regions of soybean HS genes that show 70--80% homology with the hsp22, hsp26 and the hsp70 genes of Drosophila, and these may be important in the regulation of the HS response and/or in the expression of these genes under other stress conditions. Concluding remarks Much progress has been made in our understanding of the HS response. Features of the response that are conserved across a broad spectrum of organisms include: (1) the rapid switch of the cell's transcriptional and translational machinery to the production of HS mRNAs and HS proteins, (2) the sequences of the 5' flanking regions of the HS genes which appear to regulate expression of these genes during HS, and (3) the structural features of the HS proteins which may be important for their role in thermal tolerance. However, the HS response of plants is easily distinguished from other organisms by the complexity and relative abundance of the LMW HS proteins. This is an interesting observation, but its significance is not yet known. Little information is available regarding the molecular mechanisms for the transcriptional and translational controls that mediate the HS response. Results from studies on Drosophila genes in sea urchin embryos2s and mouse cells29have shown that these introduced genes are induced by HS, indicating that there are common regulatory sequences in HS genes from diverse organisms that are recognized by the host transcriptional apparatus (for a recent review, see Ref. 6). Clearly, the HS temperature at which these foreign genes are expressed is determined by the host and is not an intrinsic property of the genes themselves. In general, the rapidity of the HS response in all organisms dictates that modification and/or cellular redistribution of critical regulatory factor(s) must occur at the onset of HS, leading to the rapid decline in translation of normal mRNAs, the immediate induction of HS gene transcription, and selective transla-
tion of the HS mRNAs. While the function of the HS proteins is also still unknown, they undoubtedly provide the basis for thermal tolerance in most organisms, and their selective localization during HS appears to be inextricably linked to the expression of this tolerance. As the ability to efficiently isolate, mutate and finally transform HS genes develops, it should be possible to construct altered HS proteins to investigate the roles of the various HS proteins in the cell. For plants, perhaps more than any other organism, the potential economic impact of heat stress on reproduction, growth, yield potential, and their responses to other, simultaneously-imposed stresses in the field, warrants a concerted effort to understand the roles of the HS genes.
Acknowledgements The authors would like to thank Drs Elizabeth Vierling, Ronald T. Nagao and J. Stephen Gantt for their helpful criticism in preparing this manuscript. This work was supported by research contracts from D O E DE AS0980ER1078 and Agrigenetics Research Associates Limited. References 1 Nover, L., Henmund, D., Neumann, D., Scharf, K-D. and Sertling, E. (1984) Biol. Zentralbl. 103, 357--435 2 Schlesinger, M. J., Aliperti, G. and Kelley, P. M. (1982) Trends Biochem. Sci. 1,222-225 3 Schlesinger, M. J., Ashburner, M. and Tissi6res, A. (eds) (1982) Heat Shock: From Bacteria to Man, Cold Spring Harbor Laboratory 4 Key, J. L., Czarnecka, E., Lin, C. Y., Kimpel, J., Mothershed, C. and Schoffl, F. (1983) in Current Topics in Plant Biochemistry and Physiology (Randall, D. D., Blevins, D. G., Larson, R. L. and Rapp, B. J., eds), Vol. 2, pp. 107-117, Univ. of Missouri-Columbia Press
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Sialic acids and their role as biological m a s k s Roland Schauer Sialic acids are a group of sugars occurring mainly as components of glycoconjugates in higher organisms. Of the many biological and pathological processes in which they participate, the regulation of molecular and cellular recognition is of outstanding importance. Structure and occurrence
The term sialic acid denotes a member of a family comprising more than 20 natural derivatives of neuraminic acid, an acid amino sugar in pyranose form with nine C-atoms (systematic name 5amino-3,5-dideoxy-D-glycero-D-galacto2-nonulopyranos-l-onic acid). Unsubstituted neuraminic acid does not occur in nature. The amino group of neuraminic acid is substituted either by an acetyl or glycoloyl residue, and the hydroxyl groups may be methylated or esterified with acetyl, lactyl, sulfate or phosphate groups 1'2 (Fig. 1). Sometimes several of these substituents are present in one sialic acid molecule. Sialic acids are the only natural sugars to show this great variety. Neuraminic acid derivatives have been found in most higher animals from the echinoderms upwards and in a few microorganisms (viruses, bacteria and protozoa) 1'2. While some cell types or animals have only one sialic acid, usually N-acetylneuraminic acid, others have several kinds. The organ with the most sialic acids known is the bovine submandibular gland, in which 14 different sialic acids have been detectedL The preferred localization of sialic acids is the outer cell membrane where these sugars often occur in high concentration and are components of glycoproteins, gangliosides or polysaccharides. The electronegative charge of a human erythrocyte, for example, is mainly due
to a dense coat of about 20 million sialic acid molecules 1. The acidic sugars are also secreted in large quantities bound to glycoproteins, e.g. serum and mucus glycoproteins, or to oligosaccharides of urine and milk 1'2. Sialic acids in flee form (not glycosidically linked) were found in larger quantities in the urine of some patients compared with healthy persons 1,3. In these macromolecular compounds (complex carbohydrates or glycoconjugates) sialic acids are a-glycosidically linked to different positions of other sugars, most frequently to galactose or N-acetylgalactosamine and rarely to Nacetylglucosamine or sialic acid itselfL2. Usually, sialic acids are found in the terminal position of oligosaccharide chains, but in gangliosides and some glycoproteins they also occur in the side position of oligosaccharide chains. In oligosialyl chains or in some echinodermal glycoconjugates, sialic acids are located within oligosaccharide chains, linking two sugar residues 1'2. These structural differences, together with the multiple forms of sialic acids, give the sialic acid part of oligosaccharide chains enormous structural diversity. The resulting biological implications are mirrored in the observation that the type of cells, their functional or developmental stages and malignancy determine the nature, linkage and density of sialic acid molecules on cell surfacesl'2'4,5.
R. Schauer is at the Biochemisches lnstitut, Christian-Albrechts-Universitiit Kiel, D-2300 Kiel, FRG.
Metabolism The pathway leading to N-acetylneuraminic acid, from which all sialic
25 Schoffi, F., Raschke, E. and Nagao, R. T. (1984) E M B O J. 3, 2491-2497 26 Cohen, R. S. and Meselson, M. (1984) Proc. Nad Acad. Sci. USA 81, 5509-5518 27 Dudler, R. and Travers, A. A. (1984) Cell 38, 391-398 28 McMahon, A. P., Novak, T. J., Britten, R. J. and Davidson, E. H. (1984) Proe. Natl Acad Sci. USA 81, 7490-7494 29 Burke, J. F. and Ish-Horowicz, D. (1982) Nucleic. Acids Res. 10, 3821-3830
acids are formed, has been well studied and extensively reviewed 1,6. However, the reactions modifying this sialic acid and leading to the various N,O-substituted neuraminic acid derivates are not known in detail and are therefore objects of intensive research. The most important reactions, their cellular localization and the enzymes involved are summarized in Fig. 2. Not much is known either about the regulation of sialic acid biosynthesis (e.g. by hormones) or the reasons for the increased sialylation and sialyltransferase activity observed in many cancer cells+. Neither can the difference in sialic acid content between X- and Y-bearing human spermatozoa 7 be explained. In sialic acid catabolism (Fig. 2), sialic acid substituents and the type of sialic acid finkage seem to influence enzymic reactions and thus the rate of degradation of sialylated glycoconjugates £,6. With the exception of sialidase from Arthrobacter ureafaciens , et(2-3)-glycosidic ReO~..... R,OHzC~ C" ~ C
R5 -CI -CH3 O -C-CH II I O OH
OH
~
0. ~
L'COOH
R4,7,8,9 -H
1t,.7.8.9)
-,c,-cM~
(~,?.8.9)
0 -C-CH-CH 3 (9) II I O OH -CH 3
{8)
-S03H -PO3H2
(8) (91
Fig. 1. Structure of natural sialic acids. N- and Osubstituents together with the position of the latter (in brackets) are indicated. These residues together with the various O-acetyl groups in one sialic acid molecule (e.g. N-acetyl-7,9-di-O-acetylneuraminic acid) give 23 known neuraminic acid derivatives 1'2. N-acetylneuraminic acid-9-phosphate is an intermediate in sialic acid biosynthesis and does not occur in glycosidic linkage.
© 1985,ElsevierSciencePublishersB.V.,Amsterdam 0376 5067185/$02.00
TIBS - September 1985
protein. As yet, there is no direct evidence that p21 functions by a cycle of GTP binding and hydrolysis, and we do not know what the relative biological effects of p21.GTP vs p21.GDP are. Nevertheless, the biochemical analysis of normal and oncogenic ras proteins has provided insight on ras function and the importance of p21 interaction with GTP. These findings indicate a direction and biochemical framework to explore the mechanism by which ras transforms mammalian cells.
7 Hughes, S. M. (1983) FEBS Lett. 164, 1--8 8 Gilman, A. G. (1984) Cell 36, 577-579 9 Stryer, L. (1983) Cold Spring Harbor Syrup. Quant. Biol. 48, 841-852 10 Dhar, R., Nieto, A., Koller, R., DeFeo-Jones, D. and Scolnick, E. M. (1984) Nucleic Acids Res 12, 3611-3618 11 Willumsen, B. M., Norris, K., Papageorge, A.G., Hubbert, N.L. and Lowy, D.R. (1984) EMBO J. 3, 2581-2585 12 Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D. and Gilman, A. G. (1984) Science 226, 860-862 13 Halliday, K. R. (1984) J. Cyclic Nucleotide and Protein Phosphorylation Res. 9, 435--448 14 Seeburg, P. H., Colby, W. W., Capon, D. J., Goeddel, D. V. and Levinson, A. D. (1984) References Nature 312, 71-75 1 Cooper, G.M. and Lane, M.A. (1983) 15 Fasano, O., Aldrich, T., Tamanoi, F., Biochim. Biophys. Acta 738, 9-20 Taparowsky, E., Furth, M. and Wiglet, M. 2 Shih, T. Y. and Weeks, M. O. (1984) Cancer (1984) Proc. Natl Acad. Sci. USA 81, Invest. 2, 109-123 4008--4012 3 DeFeo-Jones, D., Scolnick, E. M., Koller, R. 16 Temeles, G.L., Gibbs, J.B., D'Alonzo, and Dhar, R. (1983) Nature 306. 707-709 J. S., Sigal, I. S. and Scolnick, E. M. (1985) 4 Powers, S., Kataoka, T., Fasano, O., Nature 313,700-703 Goldfarb, M., Strathern, J., Broach, J. and 17 Papageorge, A., Lowy, D. and Scolnick, Wigler, M. (1984) Cell 36, 607-612 E. M. (1982)Z Virol. 44, 509-519 5 Neuman-Silberberg, F.S., Schejter, E., Hoffmann, F. M. and Shilo, B-Z. (1984) Cell 18 Finkel, T., Der, C.J. and Cooper, G. M. (1984) Cell 37, 151-158 37, 1027-1033 6 Reymond, C.D., Gomer, R.H., Mehdy, 19 Gibbs, J.B., Sigal, I.S., Poe, M. and Scolnick, E. M. (1984) Proc. Natl Acad. Sci. M.C. and Firtel, R.A. (1984) Cell 39, USA 81, 5704-5708 141-148
20 McGrath, J.P., Capon, D.J., Goeddel, D. V. and Levinson,A. D. (1984)Nature 310, 644-649 21 Mantle, V., Yamazaki, S. and Kung, H-F. (1984) Proc. Natl Acad. Sci. USA 81, 6953-6957 22 Sweet, R. W., Yokoyama, S., Kamata, T., Feramisco, J. R., Rosenberg, M. and Gross, M. (1984) Nature 311,273-275 23 Stacey, D. W. and Kung, H-F. (1984) Nature 310, 508-511 24 Feramisco, J.R., Gross, M., Kamata, T., Rosenberg, M. and Sweet, R. W. (1984) Cell 38, 109-117 25 Poe, M., Scolnick, E. M. and Stein, R. B. (1985) J. Biol. Chem. 260, 3906-3909 26 Gibbs, J. B., Ellis, R. W. and Scolnick,E. M. (1984) Proc. Natl Acad. Sci. USA 81, 2674-2678 27 Temeles, G. L., DeFeo-Jones, D., Tatchell, K., Ellinger, M. S. and Scolnick, E. M. (1984) Mol. Cell. Biol. 4, 2298-2305 28 Tamanoi, F., Walsh, M., Kataoka, T. and Wigler, M. (1984) Proc. Nag Acad. Sci. USA 81, 6924-6928 29 Kataoka, T., Powers, S., Cameron, S., Fasano, O., Goldfarb, M., Broach, J. and Wigler, M. (1985) Cell 40, 19-26 30 DeFeo-Jones, D., Tatchell, K., Robinson, L. C., Sigal, I. S., Vass, W. C., Lowy, D. R. and Scolnick, E, M. (1985) Science 228, 179-184
Heat shock in plants
exception of the genes for the 83-85 kDa class of HS proteins, none of these genes contains introns. (3) When tissues are returned to their normal growing temperature, synthesis of HS proteins ceases, synthesis of other proteins recovers gradually, and the HS m R N A s decay fairly rapidly with a halflife of less than 2 hours. (4) Heat treatments which induce HS protein synthesis also lead to the development of thermal tolerance - the ability of an organism to withstand a normally lethal temperature if it is first given a HS at a non-lethal temperature.
Janice A. Kimpel and Joe L. Key The heat shock response is a ubiquitous phenomenon in all prokaryotes and eukaryotes that have been studied. In higher plants, the general characteristics o f this response have much in c o m m o n with those o f other organisms, including a probable role in the development o f short-term thermal tolerance. A s research on the response in higher plants continues, the focus is on understanding the regulation o f the heat shock genes and the function o f the heat shock proteins.
Over the past two decades, the heat shock (HS) response has been intensively studied, first in Drosophila and subsequently in a wide diversity of animals and microorganisms (for reviews, see Refs 1-3). In the last five years, the characteristics of the HS response in plants have been investigated in many species, including maize, tomato, cotton, tobacco, carrot, Chlamydomonas, soybean, pea and millet (see Ref. 4 [and Refs therein] and Ref. 5). Thus, it is now possible to conduct a comparative analysis of this response between plants and other organisms. The HS response of plants shares J. A. Kimpel and J. L. Key are at the Department of Botany, University of Georgia, Athens, GA 30602, USA. J.A. Kimpel is currently at the Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331, USA.
many parameters with the responses to HS that have been described for Drosophila, animals and yeast: (1) Immediately following an abrupt shift of 8-10°C above the normal growing temperature, synthesis of a new group of proteins, the heat shock proteins (HS proteins) is induced and, at the same time, there is a decline in synthesis of the normal complement of cellular proteins. (2) The synthesis of these HS proteins is due to the transcription of a new set of genes, the HS genes. Many of these genes have now been sequenced, and a consensus promoter or enhancer-like sequence has been identified in the 5' flanking regions (for review, see Ref. 6). Evidence is accumulating that this core 15 bp sequence is critical to the heatinducibility of these genes. With the
Thus, the general phenotype of the HS response is highly conserved in all organisms that have been studied. This suggests that there is a fundamental role for HS proteins in cenular function during high temperature stress and possibly during recovery from the stress. However, the definition of such a function for these proteins has so far eluded researchers. We present here a concise review of recent results in research on the HS response in plants that might lead to the development of testable hypotheses about the function of these proteins and the regulation of the response.
The heat shock proteins of plants All organisms produce HS proteins that can roughly be divided into two
~) 1985.ElsevierSciencePublishersB.V. Amsterdam IB76- 5067/85/$02.00
TIBS - September 1985
354 groups: the high-molecular-weight (HMW) and the low-molecular-weight (LMW) HS proteins. In plants, the abundant HS proteins are a complex group of LMW proteins which resolve into more than 30 polypeptides during two-dimensional O'Farrell gel electrophoresis. The majority of these LMW proteins range in size from 15 to 18 kDa, but there are also HS proteins of 21, 24 and 27 kDa. The exact profile and complexity of LMW HS proteins varies among plant species, but a relatively high amount of this group of proteins is observed in all plant species4. All tissues of the plant seem to produce the full spectrum of HS proteins with the noted exceptions of pollen from Tradescant/a and maize, which do not produce any HS proteins (pp. 321-327 in Ref. 3 and Ref. 7). If this proves to be a general rule for plant pollen, it may have considerable physiological significance, particularly under field conditions. The abundance of these LMW proteins contrasts with the situation in mammalian, insect and yeast cells. In these organisms, the abundant HS proteins are HMW proteins of 65, 68, 70, 82-84, 90 and 100-110 kDa, The 70 kDa protein is by far the most abundant of all the HS proteins in these organisms, and antibodies to the protein isolated from chick cells cross-react with the 70 kDa HS proteins from Dictyostelium, Drosophila, human cell lines, and higher plants2. The HMW proteins of plants seem less complex than the LMW proteins. For a given plant species, the HMW proteins resolve into less than ten polypeptides during two-dimensional electrophoresis. There is at least one polypeptide of 90-110 kDa, one or two polypeptides of 80-90 kDa, and usually two or three polypeptides of 68-75 kDa. Heat shock proteins are synthesized very soon after organisms are placed at the HS temperature. In etiolated soybean seedlings, we have detected the synthesis of HS proteins within the first 15 min at 40°C. If these seedlings are retumed to their normal growing temperature of 28°C after between 30 rain and 2 h at 40°C, synthesis of the HS proteins declines dramatically. After 3-4 h at 28°C, synthesis of HS proteins is no longer detectable. However, the HS proteins that were made during this time are quite stable, and the majority are present in the tissue for at least 20 h8. In plants, the actual temperature at which HS proteins are maximally induced varies with each species, but it is correlated with the normal growing temperature. Pea, a cool-season species, maximally induces HS proteins around
37°(2, while millet, a warm-season grass, maximally induces HS proteins around 45°C. For maize and soybean, the optimal temperature is 40°C4. In general, synthesis of HS proteins is first detectable at temperatures about 5°C above the normal growing temperature. Synthesis of HS proteins as a percentage of total protein synthesis increases strongly as the temperature of the HS approaches the optimal temperature. Above this optimum, total protein synthesis drops off precipitously, but the majority of proteins synthesized at these higher temperatures are HS proteins. The synthesis of HS proteins results from the production of new mRNAs in the cell at the higher temperatures. In soybean seedlings, the HS mRNAs for the LMW proteins become very abundant during a 2-h HS, and this facilitated the isolation of HS cDNA clones by differential screening9. Using these cDNAs as probes, HS mRNAs can be detected within the first 5 min of a HS at 40°C. The steady state concentration of these LMW HS mRNAs increases steadily during 3-4 h of HS, then levels off and gradually declines with continued heat treatment. We do not know whether this reflects a stabilization of the HS mRNAs or a balance between synthesis and degradation of the mRNAs. In Drosophila, HS mRNAs were stable at 37°C, but they rapidly destabilized to a half-life of about 2 h when cells were returned to 25°C (Ref. 10). We have not been able to measure the half-life of the soybean HS mRNAs directly, but when seedlings are returned to 28°C, HS mRNA levels decline with an apparent half-life of 1-2 h (Ref. 8). By definition, HS proteins are a new set of proteins rapidly and abundantly produced in response to a HS, but in many organisms other stresses, such as ethanol, anoxia, arsenite, or heavy metal ions, will also induce the synthesis of HS proteins. However, with one exception, we have not detected synthesis of HS proteins in soybean seedlings under other stress conditions using incorporation of radioactive amino acids into proteins in vivo. It is possible to detect the HS mRNAs during these different stresses, but the amount of HS mRNAs is much less than that found during HS 11. Arsenite is the exception to these results; soybean seedlings incubated for 3 h in 50 ~M arsenite synthesize nearly the full spectrum of HMW and LMW HS proteins. We do not yet understand why arsenite in particular induces the HS proteins in soybean while many other stresses, which are effective in many other organisms,
do not appear to do so. Heat shock proteins and thermal tolerance From a physiological point of view, the only biological function assigned to HS proteins to date is their possible role in mediating the expression of thermal tolerance. In soybean seedlings, an absolute correlation has been found between the synthesis and accumulation of HS proteins and the ability to survive short heat treatments at otherwise lethal temperatures. It has long been known that a short period of high temperature (e.g. several minutes at 45°C, or 30 s at 50°C12), usually followed by some time at the normal growing temperature, could protect the plant from a subsequent heat treatment at an otherwise lethal temperature. We have recently demonstrated that a short HS at 45°C for 2-10 min triggers the synthesis of HS proteins. Interestingly, in these experiments, HS protein synthesis does not decline dramatically when the plants are returned to 28°C, as it does after a HS at 40°C. Instead, synthesis of HS proteins continues at high levels for approximately 2-3 h at 28°C, and then it declines gradually over the next 2-4 h. Our working hypothesis is that once the HS response is triggered, the accumulation of HS proteins is quantitatively monitored by the plant cells. When sufficient HS proteins are present in the cell to fulfil their protective function, HS protein synthesis stops. Such a hypothesis is supported by our additional observations that during prolonged heat treatment at 40°C, the synthesis of HS proteins declines after 3--4 h and is not detected after about 6 h. It is also supported by the results mentioned earlier regarding the quantitative, temperaturedependent aspects of the HS response. In some way, the organism must be able to measure its own temperature (either directly or as a differential) and mediate the extent of the HS response appropriately. It is possible that another product accumulates with the HS proteins during heat treatment which is responsible for the observed thermal tolerance. One test of this alternative hypothesis is to induce the synthesis of HS proteins without a heat treatment. This has been done in yeast using ethanol, in CHO cells using ethanol, arsenite or hypoxia (see Fig. 19 in Ref. 1 and references therein), and in soybean seedlings using arsenite '3. In all cases, thermal tolerance was observed after a pretreatment with the other stress agent. However, it is still possible to argue that factors other than
TIBS - September 1985 HS proteins are the critical determinants; perhaps stronger evidence comes from studies on mutants lacking the HS response. In Dictyostelium, a mutant was isolated which did not develop thermal tolerance to lethal temperatures when given a HS at a non-lethal temperature 14. This mutant did not synthesize the 26-32 kDa LMW HS proteins at all, and synthesis of the HMW proteins was only mildly enhanced during HS, compared to the wild-type. In E. coli, synthesis of the HS proteins is positively regulated by the htpR (hin) gene product. Mutants of E. coli lacking this gene product also fail to acquire thermal resistance during HS at a permissive temperature ~5. A double mutant of Saccharomyces cerevisiae was constructed by insertion mutagenesis at the two major 70 kDa HS protein genes~6. No synthesis of 70 kDa proteins was detectable in the mutant, yet it was as resistant as the wild-type to a brief exposure at a very high temperature after a preincubation at an intermediate temperature. Protein synthesis was still required for the acquisition of this resistance, and it was suggested that other HS proteins may be essential for protection against short-term exposure to extremely high temperatures. It is quite possible that different HS proteins have separate functions during heat treatments at various temperatures. Some HS proteins are also synthesized at specific times during development (e.g. in Drosophila) in the absence of any heat stress. It is feasible that some or all of the proteins identified as HS proteins may have additional roles in cellular function which might make selection of mutants more difficult. Another approach to understanding the role of HS proteins in thermal protection is to look at plants in the field. Plants are obligately and daily subjected to fluctuations in temperature that can be as much as 20-25°C. We have recently shown that soybean plants in the field synthesize HS mRNAs and proteins when air temperatures approach 40°C (Kimpel and Key, unpublished results). Plants in irrigated fields, which can avoid possibly deleterious effects of high air temperature by lowering their leaf temperature through evapotranspiration, have lower concentrations of HS mRNAs than nonirrigated plants. Thus, plants in the field and in the laboratory respond to transient increases in temperature by inducing the synthesis of HS proteins, and the extent of the response is determined by the severity of the heat treatment. Natural plant populations display a
355 wide range of heat tolerance, and much elegant research has focused on the physiological basis of the ability of some species to survive hot, often desert-like environments (for review, see Ref. 17). Is such heat adaptation related to HS proteins? There is as yet no research on the production of HS proteins in strongly heat tolerant species, but work on these plants indicates that other mechanisms are critical for adaptation. We hypothesize that the production of HS proteins is a short-term, induced response to heat stress at temperatures above the range of normal growing tem-~ peratures. We would predict that plants, normally adapted to grow in a very warm environment would also synthesize HS proteins at temperatures 8--10°C above the median ambient temperature. Because of the very high abundance and complexity of the LMW HS proteins of plants, it was originally suggested that these proteins might be unique to plants. The amino acid compositions of three different soybean HS proteins of about 18 kDa are over 90% homologous, but when they are compared to the 22 and 23 kDa proteins of Drosophila, the overall homology is less than 40%. However, hydropathy plots indicate four major regions of strong similarity. In the most hydrophobic domain, there is a striking conservation of sequence: Asp--Gly-Val-Leu-Thr(in the soybean genes, an Asn replaces Asp; Nagao, Czarnecka, Gudey, Schoffl and Key, unpublished results). This same sequence is found in a hydrophobic domain of the bovine a-crystallin proteinTM. This may be a clue to the function of HS proteins, since et-crystallins, major components of the vertebrate eye lens, are also LMW proteins that form large aggregates in the cells of the lens. Perhaps, LMW HS proteins also form large aggregates that perform a structural role in stabilizing various cellular constituents. In plants and Drosophila, there is evidence for such aggregation of HS proteins. In Drosophila, a 'nonnuclear structure' containing many HS polypeptides assembles and copurifies with nuclei during HS (see pp. 235-242 in Ref. 3). In tomato cell cultures and leaves, the LMW HS proteins form aggregates in the cytoplasm known as HS granules. These are only visible in the cells under HS conditions when HS proteins are presentL We have recently confirmed the existence of such large aggregates of the LMW HS proteins in soybean seedlings under HS conditions. Thus, at least one function of the LMW HS proteins could be structural. These
large aggregates in/he cell may serve as transient cellular matrices in which various cellular organelles and compartments are held and thereby stabilized. Such matrices would be maintained only during HS; once the cell temperature dropped back to normal, these aggregates would dissociate, allowing rapid recovery of normal cellular function. It is also clear that individual HS proteins become associated with specific organelles during HS. The 70 kDa protein of Drosophila concentrates in the nucleus during HS. Upon recovery from HS, this protein leaves the nucleus, but it rapidly reassociates with the nucleus during a second HS 19. In soybean seedlings, the 70 kDa HS proteins and the group of 15--18 kDa HS proteins become associated with nuclei only during HS. The 15-18 kDa HS proteins and the 21 and 24 kDa HS proteins become associated with the mitochondria during HS, but while the 15-18 kDa HS proteins dissociate from the mitochondrial fraction during recovery from HS, the 21 and 24 kDa proteins remain associated with the mitochondria after the seedlings are returned to the normal growing temperature ~3. We have recently identified another class of nuclear-encoded HS proteins which are specifically transported into chloroplasts 20. Maize, pea and soybean have major HS polypeptides in the chloroplasts that are between 23 and 25 kDa; soybean has an additional polypeptide of 27 kDa. Thus, some HS proteins associate with mitochondria during HS but do not dissociate upon recovery and some HS proteins are taken up by chloroplasts in an in vitro system. These results strongly suggest that these organelle-specific HS proteins are synthesized as precursors and processed during transport, leaving them irreversibly localized within the organeUe. In Drosophila, the localization of HS proteins is not strictly temperaturedependent, since anoxia also leads to concentration of the 70 kDa HS protein in the nucleus. However, in soybean seedlings and Drosophila 21, HS proteins synthesized during arsenite treatment are not localized to any organelle. When arsenite-treated soybean seedlings are subsequently heat-shocked, HS proteins synthesized during arsenite treatment rapidly localize with organelles, including the nuclei and mitochondria. While it seems likely that the function of HS proteins in thermal tolerance requires localization, it is not clear that such localization is a component of other physiological stresses.
356
Regulation of the HS response While many parameters of the HS response of plants have been investigated, there is little information on the mechanisms of regulation of the response. Regulation dearly occurs at both transcription and translation. Results from other organisms and our own research on soybeans indicate that during HS, normal cellular mRNAs persist in the cells but are translated very inefficiently if at all. In soybean seedlings, the level of actin mRNAs remains unchanged during HS, but the mRNA levels for the small subunit of ribulose bisphosphate carboxylase and an auxinregulated gene decrease dramatically during HS. The total poly(A)RNA content remains fairly constant during HS, and since HS mRNAs represent over 20% of the total poly(A)RNA pool after a 2-h HS, the concentrations of many normal poly(A)RNAs must decline. Thus, there must be a selective mechanism for preferential initiation and/or elongation of HS proteins on HS mRNA, relative to the population of normal mRNAs. In Drosophila, ceilfree lysates from heat-shocked cells do selectively translate HS mRNAs from a pool of total cellular mRNAs 22. When tissues are returned to normal temperatures after HS, the normal cellular mRNAs are once again efficiently translated. In soybean seedlings under prolonged heat treatment, HS mRNAs cease to be translated after about 6 h, and the translation of normal mRNAs recovers. The molecular basis for such translational regulation is not known. There has been little success in developing translation systems in vitro for more than a few plant species, so the existence of discriminating translational mechanisms has not been investigated in plants. The absence of HS mRNAs in tissues that have not been heat-shocked indicates that the induction of the HS response is obligately regulated at the level of transcription. Recently, factors have been identified that interact with RNA polymerase II and/or specific sequences in the regulatory regions of the HS genes to mediate the transcription of these genes during HS 2a.24. The identification of consensus, regulatory sequences has been the focus of much recent research (for review see Ref. 6), and we have recently completed analyses of several LMW HS genes of soybean (Ref. 25, and Nagao, R. T. et al., unpublished results). The 5' flanking regions of the soybean genes contain a sequence highly homologous to the HS consensus sequence identified in Drosophila, and it is located in a comparable
T I B S - September 1985
position to the T A T A box in the Drosophila hsp70 gene. There are additional regions of high homology to the HS consensus sequence located further upstream in the soybean LMW HS genes, and these may be important in regulating the level of expression of these genes. Recently, additional regulatory regions upstream of the HS consensus sequence have been identified that are needed for efficient promoter utilization2627. There are also numerous sequences in the 5' flanking regions of soybean HS genes that show 70--80% homology with the hsp22, hsp26 and the hsp70 genes of Drosophila, and these may be important in the regulation of the HS response and/or in the expression of these genes under other stress conditions. Concluding remarks Much progress has been made in our understanding of the HS response. Features of the response that are conserved across a broad spectrum of organisms include: (1) the rapid switch of the cell's transcriptional and translational machinery to the production of HS mRNAs and HS proteins, (2) the sequences of the 5' flanking regions of the HS genes which appear to regulate expression of these genes during HS, and (3) the structural features of the HS proteins which may be important for their role in thermal tolerance. However, the HS response of plants is easily distinguished from other organisms by the complexity and relative abundance of the LMW HS proteins. This is an interesting observation, but its significance is not yet known. Little information is available regarding the molecular mechanisms for the transcriptional and translational controls that mediate the HS response. Results from studies on Drosophila genes in sea urchin embryos2s and mouse cells29have shown that these introduced genes are induced by HS, indicating that there are common regulatory sequences in HS genes from diverse organisms that are recognized by the host transcriptional apparatus (for a recent review, see Ref. 6). Clearly, the HS temperature at which these foreign genes are expressed is determined by the host and is not an intrinsic property of the genes themselves. In general, the rapidity of the HS response in all organisms dictates that modification and/or cellular redistribution of critical regulatory factor(s) must occur at the onset of HS, leading to the rapid decline in translation of normal mRNAs, the immediate induction of HS gene transcription, and selective transla-
tion of the HS mRNAs. While the function of the HS proteins is also still unknown, they undoubtedly provide the basis for thermal tolerance in most organisms, and their selective localization during HS appears to be inextricably linked to the expression of this tolerance. As the ability to efficiently isolate, mutate and finally transform HS genes develops, it should be possible to construct altered HS proteins to investigate the roles of the various HS proteins in the cell. For plants, perhaps more than any other organism, the potential economic impact of heat stress on reproduction, growth, yield potential, and their responses to other, simultaneously-imposed stresses in the field, warrants a concerted effort to understand the roles of the HS genes.
Acknowledgements The authors would like to thank Drs Elizabeth Vierling, Ronald T. Nagao and J. Stephen Gantt for their helpful criticism in preparing this manuscript. This work was supported by research contracts from D O E DE AS0980ER1078 and Agrigenetics Research Associates Limited. References 1 Nover, L., Henmund, D., Neumann, D., Scharf, K-D. and Sertling, E. (1984) Biol. Zentralbl. 103, 357--435 2 Schlesinger, M. J., Aliperti, G. and Kelley, P. M. (1982) Trends Biochem. Sci. 1,222-225 3 Schlesinger, M. J., Ashburner, M. and Tissi6res, A. (eds) (1982) Heat Shock: From Bacteria to Man, Cold Spring Harbor Laboratory 4 Key, J. L., Czarnecka, E., Lin, C. Y., Kimpel, J., Mothershed, C. and Schoffl, F. (1983) in Current Topics in Plant Biochemistry and Physiology (Randall, D. D., Blevins, D. G., Larson, R. L. and Rapp, B. J., eds), Vol. 2, pp. 107-117, Univ. of Missouri-Columbia Press
5 Pitto, L., LoSchiavo, F., Giuliano, G. and Terzi, M. (1983) Plant Mol. Biol. 2, 231-237 6 Bienz, M. (1985) Trends Biochem. Sci. 10, 157-161 7 Cooper, P., Ho, T. H. D. and Hauptmann, R. M. (1984) Plant Physiol. 75, 431--441
8 Key, J. L., Kimpel,J. A., Vierling,E., Lin, C.Y., Nagao, R.T., Czarnecka, E. and Schoffl, F. (1985) in Changes in Eukaryotic Gene Expression in Response to Environmental Stress (Atkinson, B.G. and Walden,
D. B., eds), pp. 327-348, AcademicPress 9 Schoffl,F. and Key,J. L. (1982)J. Mol. Appl. Gen. 1, 301-314 10 DiDomanico, B. J., Bugaisky, G. E. and Lindquist, S. (1982) Proc. Natl Acad. Sci. USA 79, 6181-6185 11 Czamecka, E., Edelman, L., Schoffl, F. and Key, J. L. (1984) Plant Mol. Biol. 3, 45-58 12 Yarwood, C. E. (1961) Science 134, 941-942 13 Lin, C. Y., Roberts, J. K. and Key, J. L. (1984) Plant Physiol. 74, 152-160 14 Loomis, W. F. and Wheeler, S. A. (1982)
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36, 655--662 20 Vierling, E., Mishkind, M. L., Schmidt, G. W. and Key, J. L. (1984) J. Cell BioL 99,
342a 21 Tanguay,R. M. and Vincent,M. (1982) Can. J. B/ochem. 60, 306-315 22 Storti, R. V., Scott, M. P., Rich, A. and Pardue, M. L. (1980) Cell22, 23 Parker, C. S. and Topoi, J. (1984) Cell 37, 273-283 24 Wu, C. (1984)Nature311, 81-84
Sialic acids and their role as biological m a s k s Roland Schauer Sialic acids are a group of sugars occurring mainly as components of glycoconjugates in higher organisms. Of the many biological and pathological processes in which they participate, the regulation of molecular and cellular recognition is of outstanding importance. Structure and occurrence
The term sialic acid denotes a member of a family comprising more than 20 natural derivatives of neuraminic acid, an acid amino sugar in pyranose form with nine C-atoms (systematic name 5amino-3,5-dideoxy-D-glycero-D-galacto2-nonulopyranos-l-onic acid). Unsubstituted neuraminic acid does not occur in nature. The amino group of neuraminic acid is substituted either by an acetyl or glycoloyl residue, and the hydroxyl groups may be methylated or esterified with acetyl, lactyl, sulfate or phosphate groups 1'2 (Fig. 1). Sometimes several of these substituents are present in one sialic acid molecule. Sialic acids are the only natural sugars to show this great variety. Neuraminic acid derivatives have been found in most higher animals from the echinoderms upwards and in a few microorganisms (viruses, bacteria and protozoa) 1'2. While some cell types or animals have only one sialic acid, usually N-acetylneuraminic acid, others have several kinds. The organ with the most sialic acids known is the bovine submandibular gland, in which 14 different sialic acids have been detectedL The preferred localization of sialic acids is the outer cell membrane where these sugars often occur in high concentration and are components of glycoproteins, gangliosides or polysaccharides. The electronegative charge of a human erythrocyte, for example, is mainly due
to a dense coat of about 20 million sialic acid molecules 1. The acidic sugars are also secreted in large quantities bound to glycoproteins, e.g. serum and mucus glycoproteins, or to oligosaccharides of urine and milk 1'2. Sialic acids in flee form (not glycosidically linked) were found in larger quantities in the urine of some patients compared with healthy persons 1,3. In these macromolecular compounds (complex carbohydrates or glycoconjugates) sialic acids are a-glycosidically linked to different positions of other sugars, most frequently to galactose or N-acetylgalactosamine and rarely to Nacetylglucosamine or sialic acid itselfL2. Usually, sialic acids are found in the terminal position of oligosaccharide chains, but in gangliosides and some glycoproteins they also occur in the side position of oligosaccharide chains. In oligosialyl chains or in some echinodermal glycoconjugates, sialic acids are located within oligosaccharide chains, linking two sugar residues 1'2. These structural differences, together with the multiple forms of sialic acids, give the sialic acid part of oligosaccharide chains enormous structural diversity. The resulting biological implications are mirrored in the observation that the type of cells, their functional or developmental stages and malignancy determine the nature, linkage and density of sialic acid molecules on cell surfacesl'2'4,5.
R. Schauer is at the Biochemisches lnstitut, Christian-Albrechts-Universitiit Kiel, D-2300 Kiel, FRG.
Metabolism The pathway leading to N-acetylneuraminic acid, from which all sialic
25 Schoffi, F., Raschke, E. and Nagao, R. T. (1984) E M B O J. 3, 2491-2497 26 Cohen, R. S. and Meselson, M. (1984) Proc. Nad Acad. Sci. USA 81, 5509-5518 27 Dudler, R. and Travers, A. A. (1984) Cell 38, 391-398 28 McMahon, A. P., Novak, T. J., Britten, R. J. and Davidson, E. H. (1984) Proe. Natl Acad Sci. USA 81, 7490-7494 29 Burke, J. F. and Ish-Horowicz, D. (1982) Nucleic. Acids Res. 10, 3821-3830
acids are formed, has been well studied and extensively reviewed 1,6. However, the reactions modifying this sialic acid and leading to the various N,O-substituted neuraminic acid derivates are not known in detail and are therefore objects of intensive research. The most important reactions, their cellular localization and the enzymes involved are summarized in Fig. 2. Not much is known either about the regulation of sialic acid biosynthesis (e.g. by hormones) or the reasons for the increased sialylation and sialyltransferase activity observed in many cancer cells+. Neither can the difference in sialic acid content between X- and Y-bearing human spermatozoa 7 be explained. In sialic acid catabolism (Fig. 2), sialic acid substituents and the type of sialic acid finkage seem to influence enzymic reactions and thus the rate of degradation of sialylated glycoconjugates £,6. With the exception of sialidase from Arthrobacter ureafaciens , et(2-3)-glycosidic ReO~..... R,OHzC~ C" ~ C
R5 -CI -CH3 O -C-CH II I O OH
OH
~
0. ~
L'COOH
R4,7,8,9 -H
1t,.7.8.9)
-,c,-cM~
(~,?.8.9)
0 -C-CH-CH 3 (9) II I O OH -CH 3
{8)
-S03H -PO3H2
(8) (91
Fig. 1. Structure of natural sialic acids. N- and Osubstituents together with the position of the latter (in brackets) are indicated. These residues together with the various O-acetyl groups in one sialic acid molecule (e.g. N-acetyl-7,9-di-O-acetylneuraminic acid) give 23 known neuraminic acid derivatives 1'2. N-acetylneuraminic acid-9-phosphate is an intermediate in sialic acid biosynthesis and does not occur in glycosidic linkage.
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