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Journal of Bacteriology, May 2005, p. 3599-3601, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3599-3601.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Localization of Chaperones DnaK and GroEL in Bacterial Inclusion Bodies

M. Mar Carrió and Antonio Villaverde*

Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

Received 10 January 2005/ Accepted 31 January 2005


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ABSTRACT
 
By immunostaining and transmission electron microscopy, chaperones DnaK and GroEL have been identified at the solvent-exposed surface of bacterial inclusion bodies and entrapped within these aggregates, respectively. Functional implications of this distinct localization are discussed in the context of Escherichia coli protein quality control.


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TEXT
 
Inclusion bodies are insoluble protein aggregates frequently found in bacteria when overexpressing heterologous genes (18) whose products fail to reach the soluble, native conformation (2). A high load of unfolded or incorrectly folded polypeptides triggers conformational stress conditions under which they accumulate as amorphous particles in either the cell cytoplasm or, for secretory proteins, eventually in the periplasm (13). Aggregated proteins are not excluded from cellular control, since they can be proteolyzed in situ or removed for refolding attempts. Mechanistically, inclusion bodies can be considered reservoirs of misfolded polypeptides occurring when folding assistant cell proteins are undertitrated, and therefore their formation and disintegration are both integrated in the cellular network that surveys protein quality (6, 7). Being rather homogeneous in composition, inclusion bodies contain, apart from the recombinant product, a large set of cellular proteins (14-17). In some cases, and especially at the early stages of inclusion body formation, cellular proteins can represent up to about 50% of the total aggregate composition (4). It is generally considered that these cell proteins might be trapped through intermolecular interactions leading to aggregation or during the purification of the aggregates under low-stringency conditions (11). However, to our knowledge, possible roles of these proteins in the biology of the aggregates have not been explored. Interestingly, the chaperones DnaK and GroEL, the main drivers of Escherichia coli protein folding (together with their cochaperones DnaJ-GrpE and GroES, respectively), have been identified as components of Escherichia coli inclusion bodies (7, 12). Since they interact with misfolded, aggregation-prone polypeptide chains for folding attempts, the possibility of passive coprecipitation is totally plausible.

In this work, we have explored the localization of these chaperones in E. coli inclusion bodies formed by the aggregation-prone ß-galactosidase fusion protein VP1LAC. This protein forms large cytoplasmic inclusion bodies, and the soluble fraction remains below 50% that of total VP1LAC. The expression of the recombinant gene was induced in BL21/pJVP1LAC cultures by standard procedures as described previously (6). At 5 h after induction of gene expression, cells were harvested by centrifugation and resuspended in 0.1 M phosphate buffer (pH = 7.4) plus 4% paraformaldehyde and 0.1% glutaraldehyde. Fixed samples were included in Lowicryl and sliced in thin (90- to 100-nm) and ultra-thin (80- to 90-nm) sections for double and single immunostaining, respectively, that were deposited in Formvar-coated girds. As primary antibodies, anti-DnaK, anti-GroEL, and anti-ß-galactosidase rabbit sera were used in 0.1 M PBS (pH = 7.4) with 1% bovine serum albumin and 20 mM glycine. As secondary antibody, a gold-labeled goat anti-rabbit antibody (BBI International EMGAR-15) was used in all cases. For the double DnaK and GroEL immunostaining, the secondary goat anti-rabbit antibody (BBI International EMGAR-10) was used for distinctive GroEL detection in the opposite side of the gird. Cell structures were contrasted by using uranyl acetate and lead citrate. Samples were visualized in a JOEL 1010 electron microscope at 80 Kv.

As shown in Fig. 1, in inclusion-body-forming cells DnaK was mostly localized on the surface of the aggregates, while low labeling levels were found in the cytoplasm (Fig. 1A, B, and C). In addition, DnaK was never observed in the interior of the aggregates in any of the observed fields. On the contrary, GroEL was widely distributed by the cell cytoplasm, and no concentration on the bodies' surfaces was seen (Fig. 1D, E, and F). In contrast to DnaK, however, a significant fraction of GroEL labeling occurred within the aggregates. This distinct chaperone distribution was confirmed by the double immunostaining (Fig. 1G and H). In cells producing the parental, soluble ß-galactosidase (when fractionating, less than 5% of this protein remains attached to the insoluble cell fraction), no particular clustering was observed for any of both chaperones (Fig. 1I). The immunoreactivity of VP1LAC embedded in inclusion bodies was confirmed by ß-galactosidase staining (Fig. 1J and K), proving no constraints in the immunoreactivity of aggregated proteins.



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  		FIG. 1. In situ immunodetection of DnaK (A, B, C), GroEL (D, E, F), and ß-galactosidase (J, K) in E. coli cells producing the inclusion body-forming protein VP1LAC. Double-detection images of DnaK (15-nm gold particles) and GroEL (10-nm gold particles) on VP1LAC-producing cells (G, H) and those expressing the parental, soluble beta-galactosidase (I) are also shown. Cells incubated only with the secondary anti-rabbit antibody (BBInternational EMGAR-10) are also shown as a control of the procedure (L). Horizontal bars depicted in the panels represent 200 nm.

The exclusion of DnaK from the interior of bacterial inclusion bodies clearly indicates that the association of this chaperone with these aggregates is not due to its coprecipitation with interacting, misfolded polypeptides during their deposition. Instead, its surface-restricted localization suggests the interaction of this chaperone with already-formed aggregates. It has been described as a steady protein removal from inclusion bodies even during protein precipitation (6), and therefore the volumetric growth of these aggregates is the result of a dynamic balance between aggregation and disaggregation (7). When cellular components of the quality control system (namely, chaperones and proteases) are limiting this equilibrium is displaced towards deposition, but in the absence of de novo protein synthesis inclusion bodies are dissolved in vivo. DnaK, together with ClpB, is involved in recovering protein from thermal aggregates (19), and this chaperone is also important in in vivo inclusion body disintegration (8). The refolding activity of this chaperone would account for its presence at inclusion body's interface, where it could be actively involved in the dynamic transition observed between soluble and insoluble forms of VP1LAC (5, 10).

On the other hand, GroEL is homogenously distributed by the cytoplasm but clearly excluded from the aggregates' interface. This observation indicates that GroEL does not have direct biological activities associated to protein transfer at the body's surface or that the amount, if any, of GroEL involved is low. The presence of this chaperone inside the bodies could be due to its trapping during aggregation. Despite the fact that ß-galactosidase, because of size constrictions, cannot be encapsulated by GroEL rings (9), interactions between GroEL and ß-galactosidase derivatives have been observed both in vitro and in vivo (1, 3). In this context, the absence of a functional GroEL in a GroEL140 mutant growing at 42 C° impairs the assembling of small aggregates to form true inclusion bodies and enhances the amount of VP1LAC present in the soluble cell fraction (8). Although the positive role of GroEL in the formation of inclusion bodies could be indirect, its presence in the aggregates could also reflect its immediate involvement in the dynamic architectural organization of the aggregating proteins. Obviously, a deeper investigation of in vivo GroEL activities is required for the definitive comprehension of this chaperone as a possible modulator of protein deposition.


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ACKNOWLEDGMENTS
 
This work has been supported by grants BIO2004-0700 (MEC, Madrid, Spain) and 2002SGR-00099 (AGAUR, Barcelona, Spain) and by the Maria Francesca de Roviralta Foundation.

Electron microscopy was performed at the Scientific-Technical Services facilities of the University of Barcelona.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain. Phone: 34 935812148. Fax: 34 935812011. E-mail: avillaverde{at}servet.uab.es. Back


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Journal of Bacteriology, May 2005, p. 3599-3601, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3599-3601.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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