Ingestion of food pellets containing Escherichia coli overexpressing the heat-shock protein DnaK protects Penaeus vannamei (Boone) against Vibrio harveyi (Baumann) infection.

Feeding aquatic animals with bacterial encapsulated heat-shock proteins (Hsps) is potentially a new method to combat vibriosis, an important disease affecting aquatic animals used in aquaculture. Food pellets comprised of shrimp and containing Escherichia coli overexpressing either DnaK-DnaJ-GrpE, the prokaryotic equivalents of Hsp70-Hsp40-Hsp20, or only DnaK were fed to juveniles of the white leg shrimp Penaeus vannamei, and protection against pathogenic Vibrio harveyi was determined. Maintaining pellets at different temperatures for varying lengths of time reduced the number of live adhering E. coli, as did contact with sea water, demonstrating that storage and immersion adversely affected bacterial survival and attachment to pellets. Feeding P. vannamei with E. coli did not compromise their survival, indicating that the bacteria were not pathogenic to shrimp. Feeding P. vannamei with pellets containing bacteria overproducing DnaK (approximately 60 cells g(-1) pellets) boosted P. vannamei survival twofold against V. harveyi, suggesting that DnaK plays a role in Vibrio tolerance. Pellets containing DnaK were effective in providing protection to P. vannamei for up to 2 weeks before loss of viability and that DnaK encapsulated by these bacteria enhanced shrimp resistance against Vibrio infection.

Feeding Artemia franciscana (Kellogg) larvae with bacterial heat shock protein, protects from Vibrio campbellii infection.

Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.

Small heat shock proteins: molecular structure and chaperone function.

Small heat shock proteins (sHSPs) associate with nuclei, cytoskeleton and membranes, and as molecular chaperones they bind partially denatured proteins, thereby preventing irreversible protein aggregation during stress. sHSP monomers consist of a conserved alpha-crystallin domain of approximately 90 amino acid residues, bordered by variable amino- and carboxy-terminal extensions. The sHSPs undergo dynamic assembly into mono- and poly-disperse oligomers where the rate of disassembly affects chaperoning. The alpha-crystallin domain contains several beta-strands organized into two beta-sheets responsible for dimer formation, the basic building block of most sHSPS. The amino-terminal extension modulates oligomerization, subunit dynamics and substrate binding, whereas the flexible carboxy-terminal extension promotes solubility, chaperoning and oligomerization, the latter by inter-subunit linkage. Crystallization studies have revealed sHSP structure and function. Additionally, site-directed mutagenesis, biophysical investigations, functional studies and the discovery of relationships between mutated sHSPs and diseases have illuminated the role of sHSP within cells.

Artemin is an RNA-binding protein with high thermal stability and potential RNA chaperone activity.

Encysted embryos of the crustacean, Artemia franciscana, are among the most stress-resistant of all multicellular eukaryotes, due in part to massive amounts of p26, a small heat shock protein, that acts as a molecular chaperone. These embryos contain equally large amounts of another protein called artemin, of previously unknown function, that we report on here. Its thermal stability allows large-scale purification in about a day, using ammonium sulfate fractionation and incubation at 70 degrees C for 7 min, followed by gel filtration. The latter yields an artemin-RNA complex from which the pure protein, apo-artemin, was obtained by anion-exchange chromatography. We evaluated the possibility that artemin acts as a molecular chaperone for proteins, but obtained no evidence for that in vitro. The association of RNA with apo-artemin occurs at high temperatures and, although it is not yet clear whether artemin has a specific role as an RNA chaperone, it does bind non-polyadenylated RNAs which are then translated in vitro. Artemin-RNA is thermostable, some molecules resisting destruction after 30 min at 90 degrees C. The first order rate constant for denaturation and aggregation of artemin-RNA at 85 degrees C is 8.5 x 10(-3)min(-1), which compares well with other thermostable proteins of similar size ( approximately 500 kDa) such as the ferritins with which artemin has amino acid sequence similarity. The amount of artemin extracted from embryos that had been stored dry, under laboratory conditions, since 1951 is comparable to the amount in contemporary embryos, indicating its stability in situ, and supporting the in vitro heating studies.

Structure and function of small heat shock/alpha-crystallin proteins: established concepts and emerging ideas.

Small heat shock/alpha-crystallin proteins are defined by conserved sequence of approximately 90 amino acid residues, termed the alpha-crystallin domain, which is bounded by variable amino- and carboxy-terminal extensions. These proteins form oligomers, most of uncertain quaternary structure, and oligomerization is prerequisite to their function as molecular chaperones. Sequence modelling and physical analyses show that the secondary structure of small heat shock/alpha-crystallin proteins is predominately beta-pleated sheet. Crystallography, site-directed spin-labelling and yeast two-hybrid selection demonstrate regions of secondary structure within the alpha-crystallin domain that interact during oligomer assembly, a process also dependent on the amino terminus. Oligomers are dynamic, exhibiting subunit exchange and organizational plasticity, perhaps leading to functional diversity. Exposure of hydrophobic residues by structural modification facilitates chaperoning where denaturing proteins in the molten globule state associate with oligomers. The flexible carboxy-terminal extension contributes to chaperone activity by enhancing the solubility of small heat shock/alpha-crystallin proteins. Site-directed mutagenesis has yielded proteins where the effect of the change on structure and function depends upon the residue modified, the organism under study and the analytical techniques used. Most revealing, substitution of a conserved arginine residue within the alpha-crystallin domain has a major impact on quaternary structure and chaperone action probably through realignment of beta-sheets. These mutations are linked to inherited diseases. Oligomer size is regulated by a stress-responsive cascade including MAPKAP kinase 2/3 and p38. Phosphorylation of small heat shock/alpha-crystallin proteins has important consequences within stressed cells, especially for microfilaments.

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana.

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.

Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins.

The selective modulation of transcription exerted by steroids depends upon recognition of signalling molecules by properly folded cytoplasmic receptors and their subsequent translocation into the nucleus. These events require a sequential and dynamic series of protein-protein interactions in order to fashion receptors that bind stably to steroids. Central to receptor maturation, therefore, are several molecular chaperones and their accessory proteins; Hsp70, Hsp40, and hip modulate the 3-dimensional conformation of steroid receptors, permitting reaction via hop with Hsp90, arguably the central protein in the process. Binding to Hsp90 leads to dissociation of some proteins from the receptor complex while others are recruited. Notably, p23 stabilizes receptors in a steroid binding state, and the immunophilins, principally CyP40 and Hsp56, arrive late in receptor complex assembly. In this review, the functions of molecular chaperones during steroid receptor maturation are explored, leading to a general mechanistic model indicative of chaperone cooperation in protein folding.

Flagellar morphogenesis: protein targeting and assembly in the paraflagellar rod of trypanosomes.

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

Transcription factors and their genes in higher plants functional domains, evolution and regulation.

A typical plant transcription factor contains, with few exceptions, a DNA-binding region, an oligomerization site, a transcription-regulation domain, and a nuclear localization signal. Most transcription factors exhibit only one type of DNA-binding and oligomerization domain, occasionally in multiple copies, but some contain two distinct types. DNA-binding regions are normally adjacent to or overlap with oligomerization sites, and their combined tertiary structure determines critical aspects of transcription factor activity. Pairs of nuclear localization signals exist in several transcription factors, and basic amino acid residues play essential roles in their function, a property also true for DNA-binding domains. Multigene families encode transcription factors, with members either dispersed in the genome or clustered on the same chromosome. Distribution and sequence analyses suggest that transcription factor families evolved via gene duplication, exon capture, translocation, and mutation. The expression of transcription factor genes in plants is regulated at transcriptional and post-transcriptional levels, while the activity of their protein products is modulated post-translationally. The purpose of this review is to describe the domain structure of plant transcription factors, and to relate this information to processes that control the synthesis and action of these proteins.

The synthesis of a small heat shock/alpha-crystallin protein in Artemia and its relationship to stress tolerance during development.

Fertilized oocytes of the brine shrimp Artemia franciscana undergo either ovoviviparous or oviparous development, yielding free-swimming larvae (nauplii) or encysted gastrulae (cysts), respectively. Encystment is followed by diapause, wherein metabolism is greatly reduced; the resulting cysts are very resistant to extreme stress, including desiccation and long-term anoxia. The synthesis of p26, a small heat shock/alpha-crystallin protein produced only in oviparously developing Artemia, is shown in this paper to be transcriptionally regulated. A p26 mRNA of about 0.7 kb was detected on Northern blots in the second day after oocyte fertilization. It peaked as embryos encysted and declined rapidly when activated cysts resumed development. The appearance of p26 protein, as indicated by immunoprobing of Western blots, followed mRNA by 1 day; it also increased as encystment occurred but remained constant during postgastrula development of cysts. However, p26 underwent a marked reduction during emergence of nauplii and could not be detected in cell-free extracts of second-instar larvae. p26 entered nuclei of encysting embryos soon after synthesis and was localized therein as late as instar II, when it was restricted to a small set of salt gland nuclei. First-instar larvae derived from cysts were more thermotolerant than larvae that had developed ovoviviparously, but synthesis of p26 was not induced by heat under the experimental conditions employed. Additionally, transformed bacteria synthesizing p26 were more thermotolerant than bacteria that lacked the protein. The results support the proposal that p26, a developmentally regulated protein synthesized during embryo encystment, has chaperone activity in vivo and protects the proteins of encysted Artemia from stress-induced denaturation.

Cloning and sequencing of an alpha-tubulin cDNA from Artemia franciscana: evidence for translational regulation of alpha-tubulin synthesis.

The brine shrimp, Artemia franciscana, exhibits a limited number of tubulin isotypes which change little during early postgastrula growth. In order to better understand the synthesis of alpha-tubulins during Artemia development, a cDNA termed alphaAT1 was cloned and sequenced. Alignment analyses revealed that the polypeptide encoded by alphaAT1 is similar to alpha-tubulins from other species. Hybridization of alphaAT1 to restriction-digested DNA on Southern blots produced a simple banding pattern, indicating that Artemia have a small number of alpha-tubulin genes. Probing of Northern blots demonstrated an abundant supply of alpha-tubulin mRNA in dormant cysts, emerging nauplii and instar I larvae. However, it was not until instar I larvae were produced that the amount of polysomal alpha-tubulin mRNA increased, suggesting that synthesis of the tubulin corresponding to alphaAT1 is translationally controlled. This work provides one of the few examples where tubulin synthesis is thought to be translationally regulated. Moreover, when considered in the light of previous analyses, the findings imply that cell differentiation in postgastrula Artemia and the diversification of microtubule function certain to accompany this process occur with little or no change in alpha-tubulin composition.

Characterization of gamma-tubulin in Artemia: isoform composition and spatial distribution in polarized cells of the larval epidermis.

Microtubule arrangement is influenced by gamma-tubulin, a soluble protein of the eukaryotic cell cytosol and a component of microtubule-organizing centers. In this study, affinity purified antibodies to gamma-tubulin were prepared and their specificity demonstrated by immunostaining of Western blots and in competitive ELISAs. When employed to label mouse fibroblasts, one or two brightly stained dots appeared in each cell, a pattern characteristic of centrosomes. Antibody 9, raised to a conserved amino-terminal peptide of gamma-tubulin, was used with TU-30 (from P. Dráber) to characterize gamma-tubulin in the crustacean, Artemia franciscana. Cell-free protein extracts from Artemia contained gamma-tubulin and it purified with alpha/beta-tubulin through several preparative steps. Probing of Western blots prepared from two-dimensional gels yielded a single isoform of gamma-tubulin in Artemia with a pI of about 5.6. Immunostaining with TAT, a general antibody to alpha-tubulin, demonstrated that Artemia possess two morphological types of immune blood cells (hemocytes) with distinctive microtubule arrays. Both the compact spherical hemocytes and the flatter, spreading cells exhibited fluorescent dots, often in pairs, when labelled with antibodies to gamma-tubulin. Microtubules in polarized cells of the epidermis were also brightly stained with antibody to alpha-tubulin, revealing interphase arrangements, anastral mitotic spindles and midbodies. Antibody 9 and TU-30 gave punctate staining patterns in interphase epidermal cell layers and they occasionally labelled midbodies. Unexpectedly, gamma-tubulin was seen only rarely at both poles of mitotic spindles in epidermal cells. The complete absence of asters and the apparent lack of gamma-tubulin at all but a small number of poles indicate that formation and structure of the mitotic spindle in epidermal cells of Artemia are unusual.

Molecular characterization of a small heat shock/alpha-crystallin protein in encysted Artemia embryos.

Molecular chaperones protect cells during stress by limiting the denaturation/aggregation of proteins and facilitating their renaturation. In this context, brine shrimp embryos can endure a wide variety of stressful conditions, including temperature extremes, prolonged anoxia, and desiccation, thus encountering shortages of both energy (ATP) and water. How the embryos survive these stresses is the subject of continuing study, a situation true for other organisms facing similar physiological challenges. To approach this question we cloned and sequenced a cDNA for p26, a molecular chaperone specific to oviparous Artemia embryos. p26 is the first representative of the small heat shock/alpha-crystallin family from crustaceans to be sequenced, and it possesses the conserved alpha-crystallin domain characteristic of these proteins. The secondary structure of this domain was predicted to consist predominantly of beta-pleated sheet, and it appeared to lack regions of alpha-helix. Unique properties of the nonconserved amino terminus, which showed weak similarity to nucleolins and fibrillarins, are enrichments in both glycine and arginine. The carboxyl-terminal tail is the longest yet reported for a small heat shock/alpha-crystallin protein, and it is hydrophilic, a common attribute of this region. Site-specific differences between amino acids from p26 and other small heat shock/alpha-crystallin proteins bring into question the functions proposed for some of these residues. Probing of Southern blots disclosed a multi-gene family for p26, whereas two size classes of p26 mRNA at 0.7 and 1.9 kilobase pairs were seen on Northern blots, the larger probably representing nonprocessed transcripts. Examination of immunofluorescently stained samples with the confocal microscope revealed that a limited portion of intracellular p26 is found in the nuclei of encysted embryos and that it resides within discrete compartments of this organelle. The results in this paper demonstrate clearly that p26 is a novel member of the small heat shock/alpha-crystallin family of proteins. These data, in concert with its restriction to embryos undergoing oviparous development, suggest that p26 functions as a molecular chaperone during exposure to stress, perhaps able to limit protein degradation and thus ensure a ready supply of functional proteins when growth is reinitiated.

Molecular chaperones and the cytoskeleton.

Heat shock proteins, first observed because they are preferentially synthesized by organisms exposed to heat or other physiological stress, are also synthesized constitutively. These proteins are divided into several families, namely, HSP100, 90, 70, 60 (chaperonin), and the small heat shock/alpha-crystallin proteins. They enjoy a wide phylogenetic distribution and are important because they function as molecular chaperones, able to mediate many cellular processes through an influence on higher order protein structure. For example, molecular chaperones assist in the transport of proteins into mitochondria and chloroplasts, as well as influencing clathrin lattice dynamics, viral replication and transcriptional activation. Under conditions of stress, some molecular chaperones prevent denaturation of proteins while others may dissociate protein aggregates, refolding monomers derived therefrom or directing their proteolytic destruction. We present in this review an analysis of the emerging literature on the relationship between molecular chaperones and the cytoskeleton, a collection of polymeric structures consisting of microtubules, microfilaments and intermediate filaments. A recent development in this field is identification of the TCP-1 complex as the eukaryotic cytoplasmic chaperonin which directs folding of cytoskeletal proteins such as alpha/beta/gamma-tubulin, actin and centractin. Moreover, the TCP-1 complex is a centrosomal component, apparently involved in the nucleation of microtubules. Other molecular chaperones recognize one or more cytoskeletal elements and in most cases they modulate the assembly of and/or provide protection for their constituent proteins. For example, HSP70 protects the centrosome and perhaps intermediate filaments during heat shock, and like HSP90, it binds to microtubules. Small heat shock proteins interact with microfilaments and intermediate filaments, affect their polymerization and guard them from heat shock by a phosphorylation-dependent mechanism. We conclude that molecular chaperones have different but cooperative roles in the formation and function of the eukaryotic cell cytoskeleton.

Tubulin post-translational modifications--enzymes and their mechanisms of action.

This review describes the enzymes responsible for the post-translational modifications of tubulin, including detyrosination/tyrosination, acetylation/deacetylation, phosphorylation, polyglutamylation, polyglycylation and the generation of non-tyrosinatable alpha-tubulin. Tubulin tyrosine-ligase, which reattaches tyrosine to detyrosinated tubulin, has been extensively characterized and its gene sequenced. Enzymes such as tubulin-specific carboxypeptidase and alpha-tubulin acetyltransferase, required, respectively, for detyrosination and acetylation of tubulin, have yet to be purified to homogeneity and examined in defined systems. This has produced some conflicting results, especially for the carboxypeptidase. The phosphorylation of tubulin by several different types of kinases has been studied in detail but drawing conclusions is difficult because many of these enzymes modify proteins other than their actual substrates, an especially pertinent consideration for in vitro experiments. Tubulin phosphorylation in cultured neuronal cells has proven to be the best model for evaluation of kinase effects on tubulin/microtubule function. There is little information on the enzymes required for polyglutamylation, polyglycylation, and production of non-tyrosinatable tubulin, but the available data permit interesting speculation of a mechanistic nature. Clearly, to achieve a full appreciation of tubulin post-translational changes the responsible enzymes must be characterized. Knowing when the enzymes are active in cells, if soluble or polymerized tubulin is the preferred substrate and the amino acid residues modified by each enzyme are all important. Moreover, acquisition of purified enzymes will lead to cloning and sequencing of their genes. With this information, one can manipulate cell genomes in order to either modify key enzymes or change their relative amounts, and perhaps reveal the physiological significance of tubulin post-translational modifications.

Tubulin synthesis and assembly in differentiating neurons.

Neurons are highly polarized cells that extend long processes, the axons and dendrites, to form contacts with target cells. The formation and maintenance of this specialized morphology relies on the assembly of an organized microtubule array that is the predominant component of the neuronal cytoskeleton. During this process there is an evolution in the composition and dynamics of microtubules, resulting in stable microtubule bundles that provide structural support and function in intracellular transport along the axon. In this essay we provide an overview of the mechanisms regulating the synthesis and assembly of tubulin in differentiating neurons with particular attention to the roles of multiple tubulin isotypes, posttranslational modifications of tubulin, and microtubule-associated proteins. We conclude that, ultimately, the developmental regulation of microtubules in neurons may require the coordinated expression and posttranslational modifications of tubulin and microtubule-associated proteins to provide biochemical forms that favour specific interactions, each combination conferring distinctive dynamic and functional properties.

Purification, structure and in vitro molecular-chaperone activity of Artemia p26, a small heat-shock/alpha-crystallin protein.

Encysted brine-shrimp gastrulae bring their metabolism to a reversible standstill during diapause and quiescence, demonstrating a remarkable resistance to unfavourable environmental conditions. For example, mortality of Artemia embryos under normal temperature and hydration is very low, even after two years of anoxia, and embryos commonly experience complete desiccation as part of their developmental program. Previous evidence from our laboratories indicated that p26, an abundant low-molecular-mass cyst-specific protein capable of translocation into the nucleus, may have a protective function in Artemia cysts. p26 was purified to apparent homogeneity and a continuous sequence of 141 of its amino acids was determined by peptide sequencing, revealing that it is a member of the small-heat-shock/alpha-crystallin family of proteins. As determined by molecular-sieve chromatography and sucrose-density-gradient centrifugation, native p26 is a multimer of about 27 monomers with a molecular mass of approximately 700 kDa. Inactivation of citrate synthase was less when the enzyme was heated in the presence rather than the absence of p26. Additionally, the renaturation of heat-inactivated citrate synthase was promoted by p26. These results indicated that p26 possesses molecular-chaperone activity, a property of other small heat-shock/alpha-crystallin proteins. Our findings demonstrate that p26 has the potential to protect the macromolecular components of Artemia embryos, either as they encyst or upon exposure to environmental extremes. Protection may depend upon the ability of p26 to function as a molecular chaperone.

Production and utilization of detyrosinated tubulin in developing Artemia larvae: evidence for a tubulin-reactive carboxypeptidase.

The reversible, enzymatically driven removal and readdition of its carboxy-terminal tyrosine are major posttranslational modifications of alpha-tubulin. To study these processes isoform-specific antibodies were produced and subsequently used to characterize tyrosinated and detyrosinated tubulin in the brine shrimp, Artemia. Tyrosinated tubulin existed in relatively constant amounts on western blots of cell-free protein extracts from Artemia at all developmental stages examined, whereas detyrosinated tubulin was present after 20-24 h of postgastrula growth. In agreement with the blots, the detyrosinated isoform was observed in immunofluorescently stained larvae after 24 h of incubation, appearing first in structures of a transient nature, namely spindles and midbodies. The elongated muscle cells encircling the gut and the epithelium bordering the gut lumen were stained extensively with antibody to detyrosinated tubulin. Detyrosination was accompanied by the appearance of a tubulin-reactive carboxypeptidase, which used both nonpolymerized and polymerized tubulin as substrate. The enzyme bound to microtubules very poorly, if at all, under conditions used in this work. Several inhibitors of carboxypeptidase A had no effect on the carboxypeptidase from Artemia and revealed similarities between this enzyme and others thought to be tubulin specific. The use of inhibitors also indicated that the carboxypeptidase from Artemia recognized aspects of tubulin structure in addition to the carboxy-terminal tyrosine. Our results support the idea that detyrosinated tubulin appears in microtubules of varying stability, and they demonstrate that Artemia possess a carboxypeptidase with the potential to detyrosinate tubulin during growth of larvae.

Nuclear-cytoplasmic translocations of protein p26 during aerobic-anoxic transitions in embryos of Artemia franciscana.

Embryos of the crustacean Artemia franciscana survive continuous anoxia for periods of years, during which their metabolism comes to a reversible stand-still. A question of some interest concerns the maintenance of cellular integrity in the absence of biosynthesis and an ongoing energy metabolism. The present paper continues previous work on an abundant protein (p26) that undergoes extensive intracellular translocation during aerobic-anoxic transitions, exhibits several characteristics of stress proteins, and might be involved in metabolic regulation during aerobic-anoxic transitions. Since it has been established that intracellular pH (pHi) plays a major role in aerobic-anoxic transitions in this system we examined the pH-dependence of nuclear-cytoplasmic translocations of p26. In unincubated and aerobic-incubated embryos (pHi > or = 7.9) p26 was located in the "soluble" fraction, whereas in anoxic embryos (pH about 6.3) roughly 50% was translocated into the nucleus as shown by immunocolloidal gold electron microscopy. These nuclear translocations also took place in vitro, simply by manipulating buffer pH in a physiologically appropriate fashion. Immunostaining of Western blots prepared after two-dimensional electrophoresis revealed several isoforms of native p26. The isoelectric point of the major isoform was 7.10 +/- 0.05, a value close to the pH at which p26 translocation into the nucleus was first initiated in vitro. 31P-NMR measurements indicated that pHi was maintained at acidic levels (about 6.3) during prolonged anoxia. We also found that pHi of hydrated (0 degree C) but otherwise unincubated embryos was alkaline, allowing for rapid resumption of metabolism under permissive conditions. The significance of these pH-dependent translocations of p26 is discussed.

Interphase cells of the centric diatom, Thalassiosira fluviatilis, lack detyrosinated, nontyrosinatable and acetylated tubulin.

Within eukaryotic cells tubulin generally exists in protein families composed of closely related isoforms generated either by differential gene transcription or by posttranscriptional and posttranslational mechanisms. In this study, immunological approaches were used to examine the contribution of posttranslational modifications to tubulin heterogeneity in a centric diatom, Thalassiosira fluviatilis, and to show the spatial distribution of microtubules in these cells during their interphase. After blotting to nitrocellulose, tubulin in cell-free homogenates of T. fluviatilis was recognized by several general tubulin antibodies including one to the tyrosinated isoform, but not by antibodies to detyrosinated, nontyrosinatable nor acetylated tubulins. Immunofluorescent staining of methanol-fixed cells revealed a net-like reticulum of microtubules originating at or near the cell nucleus. For all antibodies, except one (TU-01), results obtained by immunofluorescent experiments corrobated the analysis of blotted tubulins. Furthermore, microtubules exhibited differential staining patterns corresponding to the intensity of antibody reactivity on blots. Antibody to detyrosinated tubulin, as well as TU-01, yielded a spotty pattern of fluorescence on chloroplasts. Microtubules in T. fluviatilis support normal cell function in the absence of detectable amounts of three common posttranslationally modified tubulins, perhaps due to the rigid silica frustule which maintains shape and to the absence of flagella in interphase cells.