JXB Advance Access originally published online on October 29, 2003
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Journal of Experimental Botany, Vol. 54, No. 393, pp. 2679-2689,
December 1, 2003
© 2003 Oxford University Press
Differential distribution of the cognate and heat-stress-induced isoforms of high Mr cis-trans prolyl peptidyl isomerase (FKBP) in the cytoplasm and nucleoplasm
Received 29 November 2002; Accepted 4 September 2003
,1
1 Plant Genetics Research Unit, USDA/ARS, Donald Danforth Plant Science Center, 975 N. Warson Road, St Louis, MO 63132, USA
2 Department of Plant Sciences, Tel Aviv University, Tel Aviv, Israel
* Permanent address: Department of Biology, Howard University, Washington DC, USA. To whom correspondence should be addressed. Fax: +1 314 587 1392. E-mail: eherman{at}danforthcenter.org
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Abstract |
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Wheat root tips express a 73 kDa cognate isoform and a 77 kDa heat-shock-induced isoform of peptidyl prolyl cis-trans isomerase (FK506 binding protein; FKBP) that is part of a chaperone complex with hsp90. The 73 kDa and 77 kDa FKBPs have very similar sequences, differing primarily in the N- and C-terminal 20 amino acids. In order to define the potential functional roles of these proteins, the 73 kDa and 77 kDa FKBPs were localized in root tips using antigen-affinity purified antibodies as a probe. The cognate 73 kDa FKBP is localized in the cytoplasm and appears enriched around the periphery of the early vacuole and vesicles exiting the trans-Golgi. Parallel assays with antibodies directed against tonoplast aquaporin and pyrophosphatase confirmed the association of FKBP with an early vacuole compartment. Sucrose gradient centrifugation analysis of root tip lysates also showed that 73 kDa FKBP is co-fractionated with tonoplast aquaporin and V-ATPase in a light compartment near the top of the gradient. Heat-shock treatment of root tips induces the accumulation of 77 kDa FKBP while the abundance of 73 kDa FKBP remains constant. Quantitative EM immunogold assays of the intracellular distribution of FKBP over an 8 h heat-shock time-course showed that FKBP is initially present in the cytoplasm, but is transported into the nucleus where it accumulates in the nucleoplasm and into specific subnuclear domains. The results of this study show that the intracellular distribution of the high Mr FKBPs in wheat root tips differs at normal and elevated temperatures, indicating different functional roles for the FKBP isoforms.
Key words: cis-trans prolyl isomerase, FK506-binding protein (FKBP), Golgi, nucleus, prevacuole, provacuole, vacuole.
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Introduction |
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In plants, cis-trans prolyl isomerases (PPIases) belonging to the cyclophilins, FKBPs and parvulins have been cloned (Boston et al., 1996; Landrieu et al., 2000). Cyclophilins were found in the cytosol, chloroplast and mitochondria (Gasser et al., 1990; Breiman et al., 1992; Lippuner et al., 1994; Luan et al., 1994, 1996; Fulgosi et al., 1998). The PPIases catalyse the isomerization of the peptide bond between a proline and the peptide residue. The cis-trans inter-conversion accelerated by PPIases is significant for the final protein structure because cis proline introduces bends within the protein. FK506 binding proteins (FKBPs) are found in all classes of organisms, having multiple roles in the cells, the best known being receptors for medically important immunosuppressors. Although most cellular functions of different FKBPs are still unknown, recent results obtained in plants and animals indicate that these proteins are master regulators in the control of development (for reviews see (Harar and Faure, 2001; Breiman and Camus, 2002).
Plant FK506 binding proteins (FKBP) have been isolated from several species such as wheat FKBP73 (Blecher et al., 1996; Aviezer et al., 1996), Arabidopsis 62 kDa ROF1 (Vucich and Gasser, 1996), Arabidopsis and Vicia faba FKBP15 (Luan et al., 1996), Arabidopsis pas1 encoding a 70 kDa FKBP (Vittorioso et al., 1998), maize FKBP66 (Hueros et al., 1998), and the wheat heat-stress-induced FKBP77 (Kurek et al., 1999). As in animals, the plant FKBPs are directed to different compartments in the cell.
The plant high molecular weight FKBPs differ from the small FKBP12 in that they contain one or more FKBP12- like domains, a tetratricopeptide repeat (TPR) domain and a C-terminal domain that, in most cases, binds calmodulin. They are cytosolic, showing high sequence and structural similarity among themselves and with the mammalian FKBP52 (Blecher et al., 1996; (Harar and Faure, 2001). The mammalian FKBP52, together with the hsp90, are part of the mammalian steroid receptor complex. Upon hormone binding the hsp90 soluble receptor complex dissociates and the receptors reaching the nucleus dimerize allowing them to act as transcription factors.
Most of FKBP52 localizes by indirect immunofluorescence to the nucleus with the rest being localized to microtubules in the cytoplasm (Czar et al., 1994). FKBP52 has been implicated in targeting the nuclear localization signal (NLS)-directed movement of the glucocorticoid receptor along microtubular pathways to the nucleus (Pratt, 1998). The nuclear localization signal was suggested as a potential site of immunophilin interaction with the receptor when it was noted that FKBP52 contains a conserved sequence of negatively charged amino acids complementary to the receptor nuclear localization signals (Picard and Yamamoto, 1987; Czar et al., 1995). Recently, it was found that FKBP52 binds to dynein suggesting that FKBP52 targets receptor movement toward the nucleus by determining its attachments to retrograde dynein motor protein (Silverstein et al., 1999; (Galigniana et al., 2001). Taken together these observations lead to the function of FKBP52 in targeting receptor movement (Pratt et al., 1999).
The wheat FKBP73 and wheat FKBP77 possess, close to their N-terminus, one PPIase domain with high similarity to the FKBP12, two additional FKBP12-like domains showing less sequence conservation, three TPR repeats forming the TPR domain, and a C-terminal calmodulin-binding domain. The wheat FKBPs (wFKBP) were shown to interact with HSP90 and to bind calmodulin, being able to exchange the mammalian FKBP52 in the steroid receptor complex (Reddy et al., 1998; Kurek et al., 2002).
FKBP73 and FKBP77 are highly similar proteins differing in primary sequence at the amino and carboxyl ends. The FKBP73 is expressed in immature tissues and reproductive organs, while FKBP77 is induced by heat shock in the same tissues. The close similarity of these two proteins and their expression occurring in similar tissues and developmental stages presents an opportunity to compare two closely related proteins that are differentially regulated. The presence of FKBP-binding proteins in multiple cellular compartments supports diverse roles for these proteins in both signalling and protein processing.
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Materials and methods |
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Plant material
Wheat (Triticum aestivum L.) seeds cv. Jackson were surface-sterilized and imbibed overnight. The imbibed seeds were further incubated on wet filter paper for 1 d to obtain the root tips from 2-d-old seedlings.
Anti-FKBP73 and 77 peptide antibodies
Isoform-specific antibodies for both the FKBP73 and FKBP77 were elicited by immunization with the unique carboxyterminal sequences (-CSRMTKPSAEESKA for FKBP73 and -CAKWRKTENAAGKQEAQPMASDSTA for FKBP77) peptide, produced in-house with an Applied Biosystems peptide synthesizer using standard supplier’s procedure and coupled to malamide activated haemacyanin by the added amino-terminal cysteine. The immunogen was used to inject rabbits and sera obtained on the vendor’s schedule (Spring Valley, Sykesville, MD, USA). Affinity purified anti-FKBP73 and 77 IgGs were obtained by immobilizing the peptides on an activated sepharose support (Sulfolink, Pierce Biotechnology, Rockford, IL) and passing the sera over the column. The immobilized antibodies were displaced by low pH, dialysed against TBS and quantified for concentration. The monospecific anti-FKBP73 and -FKBP77 peptide antibodies yielded only one band on SDS/PAGE immunoblots and did not exhibit any cross-reactivity with the other FKBP isoform.
One- and two-dimensional SDS/PAGE immunoblot assays and sucrose gradients
To examine the polypeptide distribution of root tip proteins after a time-course of heat stress (37 °C) treatment, 2 d whole seeds were stressed in an incubator and, at different time points, root tips were excised and ground in a mortar and pestle in 2 ml g–1 of tissue using the SDS sample buffer (Laemmli, 1970) containing 6 M urea substituting for glycerol. The samples were heated to 70 °C for 10 min and clarified by 5 min centrifugation in a microfuge. The samples were fractionated on 10% SDS/PAGE gels and blotted onto nitrocellulose membranes for antibody binding analysis as described for the sucrose gradient analysis.
For sucrose gradient analysis root tips from 2 d seedlings were extracted by gentle maceration with a mortar and pestle in 50 mM HEPES, 250 mM sorbitol, 6 mM EDTA, 1 mM DTT, and 0.1 mM PMSF pH 7.4. The cell wall fragments and other large materials were removed by centrifugation at 1000xg for 5 min at 4 °C. The resulting supernatant was loaded on a continuous 10–40% (w/v) sucrose gradient in the extraction buffer. The gradients were centrifuged overnight (14 h) in a Beckman SW-41 rotor at 39 000 rpm at 4 °C. The resulting gradient was fractionated in 0.7 ml fractions, divided in half, with one set precipitated with TCA and the other half retained frozen as replicates. The TCA pellet was washed with acetone and the pellet was solubilized in 100 µl of SDS sample buffer. SDS/PAGE fractionation of the protein pellets and electrotransfer of proteins to nitrocellulose blots were accomplished by standard procedures (Laemmli, 1970; Towbin et al., 1979). Immunological labelling of the blots was accomplished by blocking the blots in 3% gelatin in TBST and then incubating the blots overnight in a dilution of the primary antibody in 1% gelatin in TBST. The anti-FKBP serum was diluted 1:5000, the anti-
-TIP was diluted 1:10 000, the culture supernatant of V-ATPase monoclonal antibody 7A5 (gift of Dr H Sze, University of Maryland, College Park) was diluted 1:300, the anti-BiP was diluted 1:2500 (gift of Dr R Boston, North Carolina State University), and anti-yeast-hsp90 (Sigma) was diluted 1:1000. The detection of the bound antibodies was accomplished by indirect labelling with anti-IgG-peroxidase and detection by chemiluminescence. Visualization and recording of labelled bands was accomplished by exposure to Kodak OMAT X-ray film.
For floatation gradient the same procedure was followed except the initial lysate sample was made up to 45% w/v sucrose and loaded in the bottom of the tube prior to overlying the sample with the continuous sucrose gradient.
Two-dimensional gel electrophoresis and immunoblot
Two-dimensional IEF/SDS PAGE analysis was accomplished using the method of Remy and Ambard-Bretteville (1987). Lysate samples were prepared by grinding the wheat root tissue with 7 vols (w/v) of a urea/Triton X100/mercaptoethanol extraction buffer followed by centrifugation in a microfuge to remove cellular debris. The samples were fractionated with 1.5x13 cm tube gel containing wide range (pH 3.5–9.0) ampholytes at 1000 V for 2 h. The second dimension was accomplished by equilibrating with tube gel the extraction buffer containing SDS and electrophoresis as in single dimension SDS gels. The fractionated proteins were transferred to a PDVF membrane and labelled with anti--TIP antibodies as described for single dimension SDS/PAGE.
Electron microscopy and immunocytochemical assays
The 2 d root tips were fixed and embedded by the standard protocols of this laboratory (see Herman and Melroy, 1990, for detailed protocols). Briefly, this consists of fixation in 4% formaldehyde and 2% glutaraldehyde in 0.5 M phosphate buffer pH 7.4. The samples were divided in half and some of the root tips were post-fixed with 1% aqueous OsO4 for conventional EM observations while the portion was processed without osmium for use in immunocytochemical assays. The root tips were dehydrated in graded ethanol and embedded in LR White resin. For immunocytochemical assays, thin sections mounted on grids were labelled with immunoaffinity purified rabbit anti-FKBP73 antibodies, rabbit anti--TIP (a tonoplast aquaporin, provided by Dr Maarten Chrispeels, UCSD, San Diego, CA), and mouse anti-yeast-hsp90 (Sigma) diluted in TBST. Ultrathin sections of the osmicated root tips were stained with 5% aqueous uranyl acetate and 3% basic lead citrate prior to viewing.
The resulting material was visualized with a Phillips 400T electron microscope and all images were captured with an axial mounted AMT CCD camera interfaced to a Macintosh G3 computer. Images were initially captured with IP Lab spectrum software and later processed for publication with Adobe Photoshop and Illustrator.
Quantitative immunogold assays were accomplished by capturing a large number of images with the CCD camera at constant magnification. Gold particle density per unit area was determined and tested for validity by statistical analysis using analysis of variance.
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Results |
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FKBP73 has previously been shown to be present in root tips ((Aviezer et al., 1996) assayed by immunoblots using holo-FKBP73 antibodies while FKBP77 was induced by heat stress. Lysates were prepared from root tips of germinated seeds (20 °C) and after 1, 2, 4, 6, and 8 h of heat stress at 37 °C. The lysates fractionated by SDS/PAGE were transferred to PDVF membranes and parallel blots were labelled with anti-FKBP73, anti-FKBP77 and anti-hsp90 antibodies. A parallel blot was stained with amido black to confirm correct loading and concentration of protein for each time point. Using an affinity-purified FKBP73 isoform-specific antibody, a single FKBP73 band was labelled in immunoblots of SDS/PAGE fractionated root lysates, which continues to be present throughout the course of an 8 h heat stress with little apparent change in abundance (Fig. 1).
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By contrast, the FKBP77 isoform is not present at 20 °C, but is induced and accumulated during the course of 8 h of heat stress at 37 °C. The FKBP77 band appears after 2 h and a maximal level is observed after 6 h of heat stress. The weak band initially observed after 1 h of heat stress is slightly higher in Mr than the primary FKBP77 band and this band continues to be present during the 8 h time-course. Whether this band represents a modified early form of FKBP77 whose sequence possesses potential modification sites or is an immunological artefact induced by heat stress and labelled with antigen affinity purified antibodies remains unresolved and the subject for further investigations.
Hsp90 is a partner protein of both FKBP73 and FKBP77 in a cytoplasmic chaperone complex (Reddy et al., 1998). Hsp90 is highly conserved in eukaryotes and a commercially available monoclonal antibody directed against yeast hsp90 is cross-reactive with plant, animal as well as yeast hsp90. A parallel immunoblot labelled with anti-yeast-hsp90 monoclonal antibody showed a single band at 20 °C that appears to increase in concentration slightly during the heat stress. A weakly labelled band of slightly smaller Mr than hsp90 appears after 2 h of heat stress and continues to accumulate through 8 h reaching maximal apparent level after 6 h. Like the weak band observed with FKBP77 antibody labelling, it is unresolved whether this band represents a modified form of hsp90, another isoform, or an assay artefact.
At 20 °C FKBP is localized with Golgi-secretion vesicles and the prevacuole
In order to determine the intracellular distribution of FKBP73 in the root tip cells, plastic sections were labelled with recombinant FKBP73 antigen-affinity purified IgGs as the primary probe. The holo-FKBP antibody was used for all EM assays as the two antipeptide antibodies that are isoform-specific did not prove to be good probes for root tip tissue fixed and embedded in plastic. The initial examination of the intracellular labelled sites showed a punctate pattern of locules in the cytoplasm. High magnification examination of the labelled sites indicated that the gold particles were associated with small membrane vesicles associated with the enlarging immature vacuole. Figure 2 shows an example of the labelling pattern where the gold particles are associated with vesicles aggregating to form the vacuole (Fig. 2, PV). The gold particle labelling was restricted to immature vacuoles in the initial stages of assembly by vesicular aggregation and was not associated with larger, but still immature vacuoles abundant in the root tips (Fig. 2A, V). The anti-FKBP73 antibody labelled the vesicles apparently secreted from the trans-Golgi (Fig. 2A, B; G, arrows) but did not label the cisternal stack of any of the Golgi.
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The pre/early vacuole is labelled with antibodies against
-TIPThe organelles labelled by the anti-holo-FKBP73 antibody appear to be the immature vacuoles. In order to characterize and identify the vesicles that are labelled with the anti-FKBP73 antibody, antibodies elicited against the tonoplast protein
-TIP was used to label 2-d-old root tip sections. -TIP antibody was originally elicited against the major aquaporin of maturing legume seeds (Johnson et al., 1990), but subsequent experiments have shown that there are TIPs in the meristem that share an epitope(s) with the seed-specific -TIP and this antibody has proved to be a useful probe to identify tonoplast in forming vacuoles (Paris et al., 1996). The -TIP antisera yielded highly specific labelling of the membranes of the immature vacuole (Fig. 3, PV, arrows) while the vacuoles (V) forming the prevacuole were labelled less intensely.
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Sucrose gradient analysis of root tip lysate
In order to obtain correlative observations on the distribution of FKBP and with other marker proteins, wheat root tip lysates derived from 2 d germinated seeds were fractionated on continuous sucrose gradients. The resulting fractions were concentrated and analysed by SDS/PAGE-immunoblot assays with affinity-purified anti-holo-FKBP, anti-
-TIP, anti-V-ATPase, and anti-BiP as probes (Fig. 4A). The affinity purified anti-FKBP antibody labelled a 73 kDa band consisting of four fractions of low density near the top of the gradient. The additional markers for the tonoplast consisted of a polyclonal antibody directed at -TIP and a monoclonal antibody directed at the V1 sector of the V-ATPase. The -TIP antibody labelled the same low-density fraction as the FKBP. The two distinct bands observed in each fraction may be a consequence of the protein’s tendency to dimerize as a result of sucrose-induced dehydration of the membrane fractions. By contrast, the V-ATPase distribution is different from the pattern of labelling of both the anti--TIP and anti-FKBP patterns. V-ATPase is present in the low-density fractions that contain the -TIP and FKBP proteins, but the majority of V-ATPase is present in higher-density fractions as previously observed in similar gradient of oat root tips (Herman et al., 1994). An additional band was labelled with the affinity-purified anti-FKBP in the middle of the gradient. The size of this polypeptide is consistent with the expected size of the 12 kDa ER-resident cis-trans prolyl isomerase. All cis-trans prolyl isomerases share conserved sequences consisting of modules that possess the FK506 binding site and the rotamase (prolyl isomerase) active site. Probing a replicate blot with the ER resident protein BiP as a marker indicates that the weakly labelled 12 kDa FKBP-cross-reactive band is likely to be an ER protein. ER labelling was not observed in the EM-immunogold assays and this may due to a low level of apparent labelling evident on the blots that have a higher signal amplification due to the enzyme-linked assay method.
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2D gel analysis supports the presumed dimerization of TIP
The double band labelled by the
-TIP antibody could indicate either a dimerization of the protein or two distinct gene products. The dimerization of aquaporins such as TIP has been previously observed and may be induced by the dehydration of the membranes by sucrose. In order to provide an alternate form of analysis, 2 d root tips were analysed by two-dimensional gel electrophoresis using a primary extraction in the presence of 8 M urea that should impede protein aggregation artefacts. Figure 4b shows the resulting blots with parallel blots stained for total protein and a second blot labelled with the -TIP antibody. The total protein blot shows a multitude of protein spots indicating the separation technique was effective at separating the root tip polypeptides. The -TIP antibody labelled a single elongated protein band (26 kDa) that is spread by isoelectric point, but not by Mr, supporting the suggestion that the two bands, cross-reactive with anti--TIP in the sucrose gradient fractions, are a biochemical artefact.
Floatation gradient indicates FKBP73 is not tightly bound to provacuole membranes
FKBP73 and the associated cytoplasmic chaperone complex are presumed to be cytosolic proteins, yet both EM immunocytochemistry and isopycnic sucrose gradient assays indicate that FKBP73 is associated with the tonoplast membranes of the earliest stages of vacuole assembly. In order to test whether the FKBP73 is tightly associated with a membrane compartment, cellular lysates were assayed by sucrose floatation gradient. Isopycnic fractionation after loading the cellular lysate in the bottom of the centrifuge tube with 40% (w/v) resulted in all of the anti-FKBP73 antibody cross-reactivity remaining in the soluble phase and not fractionating with any membranes that would rise in the gradient (Fig. 4c).
FKBP77 accumulates in the nucleus as the consequence of heat-stress
Labelling thin sections of 37 °C heat-stressed root tips with affinity-purified anti-holo-FKBP antibody indicated that heat stress induced a different distribution of FKBP. Unlike cells grown at 20 °C where the gold label was exclusively cytoplasmic and associated with the provacuole vesicles, the nuclei of the heat-stressed root tips consistently exhibited a much higher level of gold particle label compared with the control. Figure 5A and B show an example that illustrates the intranuclear labelling of root tip nuclei of 20 °C at zero time and after 8 h at 37 °C.
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In order to characterize the accumulation of FKBP in the nucleus, quantitative immunogold analysis was conducted on a set of samples of a heat-stress time-course. Thin sections prepared from 0, 1, 2, 4, 6, and 8 h of 37 °C heat-stressed root tips were simultaneously labelled with affinity purified anti-FKBP IgGs using the same dilution, solutions and incubation time to standardize the assay. Large areas of cytoplasm and numbers of nuclei were imaged for each time point using the EM with its CCD imaging system. The gold particle density was counted per unit area and statistically valid data assembled. The resulting data are shown in Fig. 6. Comparison of the cytoplasmic and intranuclear concentration of gold particles indicate that FKBP levels rise rapidly after 37 °C heat-stress induction with the protein initially accumulating in the cytoplasm (1–4 h of heat-stress). FKBP is then gradually accumulated in the nucleus with its density exceeding that in the cytoplasm after 4 h of heat-stress.
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Double labelling indicates FKBP colocalizes with a subset of hsp90 distribution
Both FKBP73 and FKBP77 are partner proteins in the cytoplasmic chaperone complex with hsp90 (Reddy et al., 1998). Hsp90 should have a cellular distribution that overlaps that of FKBP. The nuclei from 20 °C and 37 °C heat-stressed root tip cells were examined using an anti-hsp90 monoclonal antibody and anti-FKBP IgG with double labelling assays using 5 and 10 nm gold particle sizes. Little hsp90 or FKBP was labelled in the nuclei of 20 °C cells (Fig. 7A). By contrast, in 37 °C heat-stressed cells, the nuclei contains abundant label representing sites of intranuclear hsp90 and FKBP (Fig. 7B, C).
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Discussion |
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FKBP73 and FKBP77 are highly similar proteins differing primarily in sequence differences in the amino- and carboxy-terminal domains. The results presented here indicate that there are two distinct localization sites for the cognate FKBP73 and the heat-stress-induced FKBP77, the provacuoles and nucleus, respectively. The difference in localization indicates that FKBP73 and FKBP77 have quite distinct functions involved in vacuole ontogeny at room temperature and some other intranuclear function as the consequence of heat-stress. FKBP73 and FKBP77 are constituents of the cytoplasmic chaperone complex with hsp97, hsp70 and p23 and it is likely that it is the function of this complex that is significant in these roles. The cytoplasmic chaperone complex (CCH) is a conserved feature of eukaryotic cells being found in animal and yeast cells (Pratt and Toft, 1997) as well as in plants (Pratt et al., 2001). This complex has many described and presumed roles (Pratt, 1998; Buchner, 1996) in assisting in the folding/processing of proteins and in signal transduction processes (Pratt and Toft, 1997; Fruman et al., 1994).
Most plant cells contain prominent vacuoles that occupy much of the intracellular space. The vacuole is a post-Golgi product of the secretory system that also sequesters inclusions derived from the cytoplasm and extracellular space. Vacuoles are highly dynamic organelles that are synthesized de novo in meristemic cells and are extensively modified in response to developmental regulation and environmental changes. The primary mechanism of vacuole ontogeny appears to be assembly from Golgi-derived provacuoles that fuse together producing a prevacuole that is gradually enlarged with a further hierarchy of vesicle fusions and aggregations to form the mature vacuole. The EM-immunogold results presented in this paper show that FKBP73 is associated with the provacuole vesicles and aggregates that constitute the pre/early vacuole. FKBP73 is associated only with a transient and very immature stage of vacuole ontogeny. Fractionation of root tip lysates on continuous sucrose gradients further showed that FKBP73 is associated with a low-density membrane fraction distinct from the tonoplast fraction. This low-density fraction contains an aquaporin that is cross-reactive with -TIP antibodies and the tonoplast proton pump V-ATPase. The presence of the V-ATPase in this low-density fraction from wheat root tips confirms a prior similar observation in fractions derived from oat root tips (Herman et al., 1994). However, the FKBP73 cross-reactive proteins remain in the soluble phase in a floatation gradient, indicating that FKBP73's association with the low-density membrane is labile or alternately destroyed by high concentrations of sucrose. The presence of -TIP and V-ATPase substantiates that FKBP73 is associated with a low-density tonoplast subfraction. Such subfractions could either represent membranes derived from a developmentally distinct stage of vacuole ontogeny or a subdomain of the tonoplast derived from fragmentation of vacuole. By correlating the sucrose gradients with the EM immunogold results it has been shown that this low-density fraction consists of pro/prevacuole membranes. The EM immunogold observations presented here show that the anti--TIP antibody densely labels the small provacuoles that constitute the prevacuole. This indicates that an aquaporin isoform that is immunologically cross-reactive with the -TIP group is primarily associated with the most immature stages of the vacuole formation. Identification of this aquaporin would prove a useful stage-specific probe for the formation and assembly of the vacuole in root tip cells.
The pro/prevacuole appears to be a distinct compartment that can be defined in compositional terms. This is particularly important because the morphology of prevacuole as a electron transparent vesicle bounded by a single membrane differs little from the morphology of the mature vacuole. By analogy to yeast cell vacuoles and animal cell lysosome formation the pre/provacuole should contain proteins involved in targeting and packaging secretory proteins. In plant cells the vacuolar targeting receptor BP80 (Paris et al., 1997; Ahmed et al., 1997, 2000) and cargo packaging protein AtELP are localized with the provacuoles exiting the Golgi and in the prevacuole. The presence the t-snare (AtPEP12p) involved in mediating pro/prevacuole fusion events shows that there is distinctive functional composition of the prevacuole (Sanderfoot et al., 1998, 1999). To this pro/prevacuole-specific set of proteins the high Mr cis-trans prolyl isomerase (FKBP73) and the multiprotein molecular chaperone complex can be added.
Organelle assembly is a complex process that involves regulation at many different levels including gene expression, protein synthesis and co-ordination of protein deposition, processing, and assembly. There are many stages in this process where a model would predict the involvement of extrinsic proteins and enzymes to assist in the organelle maturation. A review by Herrmann et al. (1999) outlined the concept of helper proteins as ‘outfitters, escorts and guides’ in ER lumen with a role in mediating protein trafficking to the Golgi. Each outfitter, escort and guide protein plays a specific role in assembling organelles without being part of the final structure. This concept of outfitters, escorts and guides can easily be extended to include analogous and similar proteins present in the cytoplasm as well as the mitochondria and chloroplast. Among potential partner proteins in organelle assembly are members of the various protein chaperone families. Chaperones have many established roles including folding newly synthesized proteins as well as in the repair of stress-damaged proteins. Chaperones are closely associated with several signalling pathways where their interactions could be involved in co-ordinating and regulating the process of organelle assembly. The interactions of FKBP73 with other hsps and chaperones as well as its possible function in signal transduction suggests a possible role of FKBP73 and other proteins in CCH as one potential mediator in the formation of the vacuole. The presence of 73 kDa FKBP with the initial stages of vacuole formation suggests the possibility that this complex and protein may be involved in signal and regulation processes that control vacuolation. The association of FKBP73 with the prevacuole opens new lines of inquiry on how vacuole formation and vacuolation may be controlled.
The association of FKBP77 with the nucleus provides a different perspective on the function of FKBPs and the associated CCH proteins. The possible function of FKBPs as molecular chaperones and as components of a signalling pathway could result in essentially identical activities of FKBP73 and FKBP77 being targeted at different protein partners and substrates. The function of FKBP77 is likely to be involved in some part of the damage control mechanisms that function following heat stress, with its role likely to be within the nucleus. Although FKBP77 is apparently synthesized in the cytoplasm where it transiently accumulates, it is probable that this is the synthesis and transport stages of FKBP accumulation with its stress-related function occurring after the protein enters the nucleus. The intranuclear distribution of FKBP and other stress-related proteins is likely to prove significant in regulating and preserving transcriptional capability in response to abiotic stress. The partner proteins and substrates for both FKBP73 and FKBP77 may prove to be significant in vacuole ontogeny and in the heat-stress response.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ahmed SU, Bar-Peled M, Raikhel NV. 1997. Cloning and subcellular location of an Arabidopsis receptor-like protein that shares common features with protein-sorting receptors of eukaryotic cells. Plant Physiology 114, 325–336.[Abstract]
Ahmed SU, Rojo E, Kovaleva V, Venkataraman S, Dombrowski JE, Matsuoka K, Raikhel NV. 2000. The plant vacuolar sorting receptor AtELP is involved in transport of NH(2)-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana. Journal of Cell Biology 149, 1335–1344.
Aviezer K, Blecher O, Erel N, Callebaut I, Aviezer K, Breiman A. 1996. A novel wheat peptidyl prolyl cis trans isomerase: cDNA isolation, structure, enzymatic activity, and expression. Plant Molecular Biology 32, 493–504.[CrossRef][ISI][Medline]
Blecher O, Erel N, Callebaut I, Aviezer K, Breiman A. 1996. A novel wheat peptidyl- prolyl cis trans isomerase: cDNA isolation, structure, enzymatic activity, and expression. Plant Molecular Biology 32, 493–504.[CrossRef][ISI][Medline]
Boston RS, Viitaner PV, Vierling E. 1996. Molecular chaperones and protein folding in plants. Plant Molecular Biology 32, 191–222.[CrossRef][ISI][Medline]
Breiman A, Fawcett TW, Ghirardi ML, Mattoo AK. 1992. Plant organelles contain distinct peptidyl-prolyl-cis-trans-isomerases. Journal of Biological Chemistry 267, 21293–21296.
Breiman A, Camus I. 2002. The involvement of mammalian and plant FK506 binding proteins (FKBPs) in development Transgenic Research 11, 373–379.[CrossRef][ISI][Medline]
Buchner J. 1996. Supervising the fold: functional principles of molecular chaperones. FASEB Journal 10, 10–19.[Abstract]
Czar MJ, Lyons RH, Welsh MJ, Renoir JM, Pratt WB. 1995. Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus. Molecular Endocrinology 9, 1549–1560.[Abstract]
Czar MJ, Owens Grillo JK, Yem Aw, Leach KL, Deibel MR, Welsh MJ, Pratt WB. 1994. The hsp56 immunophilin component of untransformed steroid receptor is localized both to microtubules in the cytoplasm and the same non-random regions within the nucleus as the steroid receptor. Molecular Endocrinology 8, 1731–1741.[Abstract]
Fulgosi H, Vener, AV, Altschmied L, Herrmann, RG, Andersson B. 1998. A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen. EMBO Journal 17, 1577–1587.[CrossRef][ISI][Medline]
Fruman DA, Burakoff SJ, Bierer BE. 1994. Immunophilins in protein folding and immunosuppression. FASEB Journal 8, 391–400.[Abstract]
Galigniana MD, Radanyi C, Renoir JM, Housley PR, Pratt WB. 2001. Evidence that the peptidylprolyl isomerase domain of the hsp90-binding immunophilin FKBP52 is involved in both dynein interaction and glucocorticoid receptor movement to the nucleus. Journal of Biological Chemistry 276, 14884–14889.
Gasser CH, Gunning DA, Budelier KA, Brown SM. 1990. Structure and expression of cytosolic cyclophilin /peptidyl prolyl cis/trans isomerase of higher plants and production of active tomato cyclophilin in E. coli. Proceedings of the National Academy of Sciences, USA 87, 9519–9523.
Harar Y, Faure JD. 2001. Plant FKBPs at the crossroads of folding and transduction pathways. Trends in Plant Science 6, 426–431.[CrossRef][ISI][Medline]
Herman EM, Melroy DL. 1990. Electron microscopic immunocytochemistry in plant molecular biology. In. Plant molecular biology manual, B13. The Hague, The Netherlands: Kluwer Academic Publishers, 1–24.
Herman EM, Li H, Su R, Larsen P, Hsu HT, Sze H. 1994. Vacuolar-type H+-ATPases are associated with the endoplasmic reticulum and provacuoles of root tip cells. Plant Physiology 106, 1313–1324.[Abstract]
Herrmann JM, Malkus P, Schekman R. 1999. Out of the ER—outfitters,escorts and guides. Trends in Cell Biology 9, 15–17.[CrossRef][ISI][Medline]
Hueros G, Rahfeld J, Salamini F, Thompson R. 1998. A maize FK506-sensitive immunophilin, mzFKBP-66, is a peptidylproline cis-trans-isomerase that interacts with calmodulin and a 36 kDa cytoplasmic protein. Planta 205, 121–131.[CrossRef][ISI][Medline]
Johnson KD, Hofte H, Chrispeels MJ. 1990. An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GlpF). The Plant Cell 2, 525–532.
Kurek I, Aviezer K, Erel N, Herman E, Breiman A. 1999. The wheat peptidyl prolyl cis trans isomerase is heat induced and developmentally regulated. Plant Physiology 119, 693–704.
Kurek I, Dulberger R, Azem A, BenTzvi B, Sudhakar D, Christou P, Breiman A. 2002. Deletion of the C terminal 138 amino acid of wheat FKBP73 abrogates calmodulin binding, dimerisation and male fertility in transgenic rice. Plant Molecular Biology 48, 369–381.[CrossRef][ISI][Medline]
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Landrieu I, De Veylder L, Fruchart LS, Odaert B, Casteels P, Portetelle D, Van Montagu M, Inzé D, Lippens G. 2000. The Arabidopsis thaliana Pin1At gene encodes a single domain phosphorylation-dependent peptidyl prolyl cis/trans isomerase. Journal of Biological Chemistry 275, 10577–10581.
Lippuner V, Chou I, Scott SV, Ettinger WF, Theg SM, Gasser CH. 1994. Cloning and characterization of chloroplast and cytosolic forms of cyclophilin from Arabidopsis thaliana. Journal of Biological Chemistry 269, 7863–7868.
Luan S, Albers MW, Schreiber SL. 1994. Light-regulated, tissue-specific immunophilins in higher plant. Proceedings of the National Academy of Sciences, USA 91, 984–988.
Luan S, Kudla J, Gruissem W, Schreiber SL. 1996. Molecular characterization of a FKBP-type immunophilin from higher plants. Proceedings of the National Academy of Sciences, USA 93, 6964–6969.
Paris N, Rogers SW, Jiang L, Kirsch T, Beevers L, Phillips TE, Rogers JC. 1997. Molecular cloning and further characterization of a probable plant vacuolar sorting. Plant Physiology 115, 29–39.[Abstract]
Paris N, Stanley MC, Jones RL, Rogers JC. 1996. Plant cells contain two functionally distinct vacuolar compartments. Cell 85, 563–572.[CrossRef][ISI][Medline]
Picard D, Yamamoto KR. 1987. Two signals mediate hormone dependent nuclear localization of the glucocorticoid receptor. EMBO Journal 6, 3333–3340.[ISI][Medline]
Pratt WB. 1998. The hsp90-based chaperone system:involvement in signal transduction from a variety of hormone and growth factor receptor. Proceedings of the Society for Experimental Biology and Medicine 217, 420–434.[Abstract]
Pratt WB, Krishna P, Olsen LJ. 2001. Hsp90-binding immunophilins in plants: the protein movers. Trends Plant Science 6, 54–58.[CrossRef][ISI][Medline]
Pratt WB, Silverstein AM, Galigniana MD. 1999. A model for the cytoplasmic trafficking of signalling proteins involving the hsp90-binding immunophilins and p50cdc37. Cell Signal 11, 839–851.[CrossRef][ISI][Medline]
Pratt WB, Toft DO. 1997. Steroid receptor interactions with heat shock protein and immunophilin chaperones. Endocrinology Reviews 18, 306–360.
Reddy RK, Kurek I, Silverstein AM, Chinkers M, Breiman A, Krishna P. 1998. High-molecular-weight FK506-binding proteins are components of heat-shock protein 90 heterocomplexes in wheat germ lysate. Plant Physiology 118, 1395–1401.
Remy R, Ambard-Bretteville F. 1987. Two dimensional electrophoresis in the analysis and preparation of cell organelle polypeptides. Methods in Enzymology 148, 623–632.
Sanderfoot AA, Ahmed SU, Marty-Mazars F, Rapoport I, Kirchhausen T, Marty F, Raikhel NV. 1998. A putative vacuolar cargo receptor partially colocalizes with AtPEP12p on a prevacuolar compartment in Arabidopsis roots. Proceedings of the National Academy of Sciences, USA 95, 9920–9925.
Sanderfoot AA, Kovaleva V, Zheng H, Raikhel NV. 1999. The t-SNARE AtVAM3p resides on the prevacuolar compartment in Arabidopsis root cells. Plant Physiology 121, 929–938.
Silverstein AM, Galigniana MD, Kanelakis KC, Radanyi C, Renoir JM, Pratt WD. 1999. Different regions of the immunophilin FKBP52 determine its association with the glucocorticoid receptor hsp90and cytoplasmic dynein. Journal of Biological Chemistry 274, 36980–36986.
Towbin H, Staehelin T, Gordon J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350–4354.
Vittorioso P, Cowling R, Faure JD, Caboche M, Bellini C. 1998. Mutation in the Arabidopsis PASTICCINO1 gene, which encodes a new FK506-binding protein-like protein, has a dramatic effect on plant development. Molecular and Cellular Biology 18, 3034–3043.
Vucich VA, Gasser CS. 1996. Novel structure of a high molecular weight FK506 binding protein from Arabidopsis thaliana. Molecular and General Genetics 252, 510–517.
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