Intracellular localization of a group II chaperonin indicates a membrane-related function

Intracellular localization of a group II chaperonin indicates a membrane-related function
October 20, 2003 (received for review August 14, 2003)
Published online before print December 12, 2003
Jonathan D. Trent * , Hiromi K. Kagawa , Chad D. Paavola *, R. Andrew McMillan *, Jeanie Howard , Linda Jahnke *, Colleen Lavin , Tsegereda Embaye , and Christopher E. Henze *
PNAS | December 23, 2003
*National Aeronautics and Space Administration Ames Research Center, Moffett Field, CA 94035; SETI Institute, 2035 Landings Drive, Mountain View, CA 94043; and Integrated Microscopy Resource, University of Wisconsin, 1975 Observatory Drive, Madison, WI 53706
Communicated by Samuel Karlin, Stanford University, Stanford, CA,
Chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. We observed, however, that the group II chaperonins known as rosettasomes in the hyperthermophilic archaeon Sulfolobus shibatae, are not cytoplasmic but membrane associated. This association was observed in cultures grown at 60¨?C and 76¨?C or heat-shocked at 85¨?C by using immunofluorescence microscopy and in thick sections of rapidly frozen cells grown at 76¨?C by using immunogold electron microscopy. We observed that increased abundance of rosettasomes after heat shock correlated with decreased membrane permeability at lethal temperature (92¨?C). This change in permeability was not seen in cells heat-shocked in the presence of the amino acid analogue azetidine 2-carboxylic acid, indicating functional protein synthesis influences permeability. Azetidine experiments also indicated that observed heat-induced changes in lipid composition in S. shibatae could not account for changes in membrane permeability. Rosettasomes purified from cultures grown at 60¨?C and 76¨?C or heat-shocked at 85¨?C bind to liposomes made from either the bipolar tetraether lipids of Sulfolobus or a variety of artificial lipid mixtures. The presence of rosettasomes did not significantly change the transition temperature of liposomes, as indicated by differential scanning calorimetry, or the proton permeability of liposomes, as indicated by pyranine fluorescence. We propose that these group II chaperonins function as a structural element in the natural membrane based on their intracellular location, the correlation between their functional abundance and membrane permeability, and their potential distribution on the membrane surface.
Nearly all organisms respond to heat and other stresses by synthesizing a small subset of proteins known as heat shock, or stress, proteins (HSPs), the production of which correlates with an increased tolerance for lethal conditions (1, 2). There is compelling evidence that HSPs participate in this so-called acquired tolerance (3ǃÏ5), although which HSPs are critical and how they function remains a topic of active research and seems to differ for different organisms or cell types (6). In the hyperthermophilic archaeon Sulfolobus shibatae, which grows optimally at 83¨?C (7), acquired thermotolerance at lethal temperatures (>90¨?C) correlates with the increased synthesis of primarily two 60-kDa HSPs known as TF55 and (8ǃÏ10). These proteins are isolated from cells as subunits of double-ring complexes called rosettasomes that reportedly share structural and functional features with chaperonins (11).
Sequence comparisons of chaperonin subunits and structural analyses of the double rings have led to the recognition of two groups of chaperonins (12). The so-called group I chaperonins found in bacteria and the chloroplasts and mitochondria of Eukarya are composed of identical or two closely related subunits arranged in two stacked rings with seven subunits each. The group II chaperonins found in Archaea and Eukarya are composed of identical or diverse subunits arranged in rings of eight or nine subunits. Chaperonins in both groups are reported to play a role in refolding stress-damaged proteins or folding newly synthesized proteins in vivo (for review see ref. 13).
The hypothesis that protein folding is the primary function of all chaperonins stems from observations that they are all composed of 60-kDa HSPs (HSP60s), heat and other HSP-inducing stresses are known to unfold proteins (14), and for group I chaperonins there is evidence for folding or refolding proteins (13). For archaeal group II chaperonins, although it has been demonstrated that they are able to recognize and bind to unfolded proteins (11) and in some cases promote their refolding in vitro (15, 16), there is growing evidence they may have other functions in vivo (17ǃÏ20).
To explore the role of group II chaperonins in vivo, we investigated the intracellular location of rosettasomes in S. shibatae under normal, heat, and cold shock conditions. We reasoned that if rosettasomes are involved in protein folding they should be located in the cytoplasm where most protein folding is presumed to occur. We observed, however, that rosettasomes are membrane associated under all conditions. We therefore investigated whether changes in their abundance correlate with changes in membrane permeability, whether they bind to lipid components of the S. shibatae or model membranes, and whether their presence on liposomes influences the structure of the lipids or their permeability to protons. Based on our observations, we hypothesize that these group II chaperonins function as a membrane "skeleton" in S. shibatae that interacts with lipids and/or membrane-associated proteins to impact the permeability and stability of the cytoplasmic membrane.
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