Purification, cDNA cloning and Northern‐blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons

Tsugeki, Ryuji; Mori, Hitoshi; Nishimura, Mikio

doi:10.1111/j.1432-1033.1992.tb17309.x

Cited by 48 publications

(31 citation statements)

References 38 publications

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“…Previously reported N-terminal sequences identified for Hsp60 isolated from mitochondria of pumpkin (Cucurbita sp., GenBank accession no. X70868) (28) and human (Homo sapiens, GenBank accession no. P10809) (29) and the presequences of these proteins, as well as the N terminus of E. coli Hsp6O (GenBank accession no.…”

Section: P17203 P17204mentioning

confidence: 99%

A common evolutionary origin for mitochondria and hydrogenosomes.

Bui

Bradley

Johnson

1996

Proc. Natl. Acad. Sci. U.S.A.

291

152

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Trichomonads are among the earliest eukaryotes to diverge from the main line of eukaryotic descent. Keeping with their ancient nature, these facultative anaerobic protists lack two "hallmark" organelles found in most eukaryotes: mitochondria and peroxisomes. Trichomonads do, however, contain an unusual organelle involved in carbohydrate metabolism called the hydrogenosome. Like mitochondria, hydrogenosomes are double-membrane bounded organelles that produce ATP using pyruvate as the primary substrate. Hydrogenosomes are, however, markedly different from mitochondria as they lack DNA, cytochromes and the citric acid cycle. Instead, they contain enzymes typically found in anaerobic bacteria and are capable of producing molecular hydrogen. We show here that hydrogenosomes contain heat shock proteins, Hsp7O, Hsp6O, and HsplO, with signature sequences that are conserved only in mitochondrial and a-Gram-negative purple bacterial Hsps. Biochemical analysis of hydrogenosomal Hsp6O shows that the mature protein isolated from the organelle lacks a short, N-terminal sequence, similar to that observed for most nuclear-encoded mitochondrial matrix proteins. Moreover, phylogenetic analyses of hydrogenosomal Hsp7O, Hsp6O, and HsplO show that these proteins branch within a monophyletic group composed exclusively of mitochondrial homologues. These data establish that mitochondria and hydrogenosomes have a common eubacterial ancestor and imply that the earliest-branching eukaryotes contained the endosymbiont that gave rise to mitochondria in higher eukaryotes.

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Section: P17203 P17204mentioning

confidence: 99%

A common evolutionary origin for mitochondria and hydrogenosomes.

Bui

Bradley

Johnson

1996

Proc. Natl. Acad. Sci. U.S.A.

291

152

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“…Eukaryotic representatives of this group include the chloroplast Rubisco subunit-binding protein (Hemmingsen and Ellis, 1986;Hemmingsen et al, 1988;Martel et al, 1990;Madueñ o et al, 1993) and the mitochondrial cpn60 protein (Prasad and Hallberg, 1989;Tsugeki et al, 1992). The maize and A. thaliana mitochondrial cpn60 genes have been isolated and found to be encoded in the nucleus (Prasad and Stewart, 1992).…”

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confidence: 99%

Heat-Stress Response of Maize Mitochondria1

et al. 1998

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We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42°C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a ؉13°C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heatstress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heatshock protein superfamily.

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“…Although certain bacteria and mitochondria also possess multiple genes for cpn60 (24,34,35), apart from plastids there are no reports of purified cpn60 14-mers that contain two divergent subunits. Likewise, immunological studies indicate that the double domain cpn10 is restricted to photosynthetic eucaryotes (25,33).…”

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confidence: 99%

Reconstitution of Higher Plant Chloroplast Chaperonin 60 Tetradecamers Active in Protein Folding

Dickson¹,

Weiss²,

Howard³

et al. 2000

Journal of Biological Chemistry

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Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, ␣ and ␤. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the ␣ and ␤ subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the ␤ subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, ␣ subunits only assemble in the presence of ␤ subunits. Although ␤ and ␣/␤ 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted ␣/␤ 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10. In contrast, the folding reaction mediated by the reconstituted ␤ 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.

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Purification, cDNA cloning and Northern‐blot analysis of mitochondrial chaperonin 60 from pumpkin cotyledons

Cited by 48 publications

References 38 publications

A common evolutionary origin for mitochondria and hydrogenosomes.

A common evolutionary origin for mitochondria and hydrogenosomes.

Heat-Stress Response of Maize Mitochondria1

Reconstitution of Higher Plant Chloroplast Chaperonin 60 Tetradecamers Active in Protein Folding

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