Expression and subcellular localization of cpn60 protein family members in Leishmania donovani

Schlüter, Anne; Wiesgigl, Martina; Hoyer, Cornelia; Fleischer, Sandra; Klaholz, Linda; Schmetz, Christel; Clos, Joachim

doi:10.1016/s0167-4781(00)00028-2

Cited by 44 publications

(47 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In studies reported here, the HSP60s in S. shibatae, which form rosettasomes, are shown to be membrane associated. There are previous reports that HSP60s in bacteria and mitochondria, which form group I chaperonins, are membrane associated (33)(34)(35)(36)(37). There are also reports that the HSP60-related TCP1s in eukarya, which form group II chaperonins (known as CCT or TriC), are membrane associated (38)(39)(40)(41).…”

Section: Discussionmentioning

confidence: 99%

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

Trent

Kagawa

Paavola

et al. 2003

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

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Trent

Kagawa

Paavola

et al. 2003

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

View full text Add to dashboard Cite

show abstract

“…A recombinant protein comprising 125 amino acids (Leu-259 to Phe-375) of P. falciparum PfBiP (35) was produced in Escherichia coli from the plasmid vector pJC45 (36) and used for the immunization of a rabbit. The specificity of the serum was tested by immunoblotting.…”

Section: Methodsmentioning

confidence: 99%

Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes

Burghaus

Lingelbach

2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a ratelimiting step.Plasmodium falciparum, the parasite that causes the most severe form of malaria, spends part of its life cycle in human erythrocytes. Here it resides within the so-called parasitophorous vacuole, which is bound by the parasitophorous vacuolar membrane (PVM).1 The vacuole constitutes a separate compartment in the infected red blood cell (iRBC) that is distinct from the cytosol of the parasite and from the cytosol of the erythrocyte, respectively (1). Most proteins secreted from P. falciparum are not released into an extracellular space but are transported to various destinations within the iRBC: the parasitophorous vacuole or the erythrocyte cytosol. Membranebound proteins are found in the PVM and erythrocyte plasma membrane (2, 3). It is completely unknown which information within a polypeptide chain determines whether a protein is transported across the PVM. Recently we described experimental evidence for a transport pathway that involves the release of secreted parasite proteins into the vacuolar space and translocation across the PVM in a subsequent step (4). This model infers that a sorting mechanism must operate within the vacuole that discriminates between vacuolar resident proteins and proteins destined for a location beyond the PVM (5). Sorting could involve two different principles: the retention of vacuolar proteins or, alternatively, the recognition of protein signals that mediate translocation across the PVM.A widely used experimental approach for the identification of protein targeting and sorting signals in eukaryotic cells is the fusion of putative signal sequences to reporter proteins and their subsequent localization in the transfected cell. Although...

show abstract

“…In contrast to the model organisms investigated so far in this context, the kinetoplastida do not belong to the Eukaryote Crown Group. Comparative sequence analyses group them with the Euglenozoa, which must have branched off very early in the evolution of the eukaryota (Schlueter et al, 2000). Nevertheless, as shown in this article, Hsp90 is used in a role very similar to its homologues in metazoan organisms.…”

Section: A Role For Hsp90 In Posttranscriptional Stress Responsementioning

confidence: 96%

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

Wiesgigl

Clos

2001

MBoC

Self Cite

185

174

View full text Add to dashboard Cite

The differentiation of Leishmania parasites from the insect stage, the promastigote, toward the pathogenic mammalian stage, the amastigote, is triggered primarily by the rise in ambient temperature encountered during the insect-to-mammal transmission. We show here that inactivation of heat shock protein (Hsp) 90, with the use of the drugs geldanamycin or radicicol, mimics transmission and induces the differentiation from the promastigote to the amastigote stage. Geldanamycin also induces a growth arrest of cultured promastigotes that can be forestalled by overexpression of the cytoplasmic Hsp90. Moreover, we demonstrate that Hsp90 serves as a feedback inhibitor of the cellular heat shock response in Leishmania. Our results are consistent with Hsp90 homeostasis serving as cellular thermometer for these primitive eukaryotes, controlling both the heat shock response and morphological differentiation. INTRODUCTIONProtozoan parasites of the genus Leishmania are transmitted by blood-feeding sand flies to mammalian hosts, including humans, whereby they encounter a rise in ambient temperature. Subjecting cultured insect stages, the promastigotes, of various Leishmania species to a similar temperature upshift in vitro results in the transient posttranscriptional upregulation of both heat shock protein (Hsp) 90 (also known as Hsp83) and Hsp70 synthesis and a persistent induction of Hsp100 expression Brandau et al., 1995;Krobitsch et al., 1998). Moreover, species such as L. mexicana, L. pifanoi, and L. donovani will, during heat stress and acidification of the medium, show a morphological transition toward the mammalian stage, the amastigote (Zilberstein and Shapira, 1994;Saar et al., 1998). Such axenic amastigotes are indistinguishable from true host tissue-derived intracellular amastigotes (Bates, 1993;Pan et al., 1993;Charest and Matlashewski, 1994;Charest et al., 1996;Gupta et al., 1996;Saar et al., 1998). Thus, the rise of temperature encountered during transmission can be viewed not as stress but rather as signal for cellular differentiation.We have shown previously that Hsp100 is critical for the expression of some amastigote stage-specific proteins and for overall virulence of the parasites (Hubel et al., 1997;Krobitsch et al., 1998); however, gene replacement mutants lacking Hsp100 still show, induced by elevated temperature, morphological differentiation to viable amastigote-like culture stages, indicating that the induction of morphological differentiation occurs independently or upstream of Hsp100 (Krobitsch et al., 1998;Krobitsch and Clos, 1999).Another chaperone, Hsp90 (Hsp83), is one of the most abundant proteins in Leishmania, constituting 2.8% of the cellular protein . Hsp90 (Hsp83) is encoded by multiple gene copies and is found in the soluble fraction of the cytoplasm (Shapira and Pinelli, 1989;Brandau et al., 1995;Hubel and Clos, 1996). Its sequence is distinct from the Grp94 homologue of L. infantum, which was characterized recently (Larreta et al., 2000). In higher eukaryotes and in yeast, cytosolic H...

show abstract

Expression and subcellular localization of cpn60 protein family members in Leishmania donovani

Cited by 44 publications

References 34 publications

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

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

Luciferase, When Fused to an N-terminal Signal Peptide, Is Secreted from Transfected Plasmodium falciparum and Transported to the Cytosol of Infected Erythrocytes

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

Contact Info

Product

Resources

About