In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation.

The transport of proteins into the nucleus is a receptor-mediated process that is likely to involve between 50-100 gene products, including many that comprise the nuclear pore complex. We have developed an assay in Saccharomyces cerevisiae for the nuclear transport of green fluorescent protein fused to the SV-40 large T antigen nuclear localization signal (NLS-GFP). This assay allows the measurement of relative NLS-GFP nuclear import rates in wild-type and mutant cells under various physiological conditions. Probably the best understood component of the nuclear transport apparatus is Srp1p, the NLS receptor, which binds NLS-cargo in the cytoplasm and accompanies it into the nucleus. When compared to SRP1+ cells, NLS-GFP import rates in temperature-sensitive srp1-31 cells were slower and showed a lower temperature optimum. The in vivo transport defect of the srp1-31 cells was correlated with the purified protein's thermal sensitivity, as assayed by in vitro NLS peptide binding. We show that the kinetics of NLS-directed nuclear transport in wild-type cells is stimulated by the elevated expression of SSA1, which encodes a cytoplasmic heat shock protein 70 (Hsp70). Elevated Hsp70 levels are sufficient to suppress the NLS-GFP import defects in srp1-31 and nup82-3 cells. NUP82 encodes a protein that functions within the nuclear pore complex subsequent to docking. These results provide genetic evidence that Hsp70 acts during both targeting and translocation phases of nuclear transport, possibly as a molecular chaperone to promote the formation and stability of the Srp1p-NLS-cargo complex.

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The Heat Shock Protein Ssa2p Is Required for Import of Fructose-1,6-Bisphosphatase into Vid Vesicles

The Journal of Cell Biology ◽

10.1083/jcb.150.1.65 ◽

2000 ◽

Vol 150 (1) ◽

pp. 65-76 ◽

Cited By ~ 51

Author(s):

C. Randell Brown ◽

Jameson A. McCann ◽

Hui-Ling Chiang

Keyword(s):

Saccharomyces Cerevisiae ◽

Heat Shock ◽

Heat Shock Protein ◽

Binding Proteins ◽

The Other ◽

Atp Binding ◽

Wild Type ◽

Fructose 1,6 Bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Δssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Δssa1, Δssa3, or Δssa4 strains. Likewise, in vitro import was impaired for the Δssa2 strain, but not for the other Δssa strains. The cytosol was identified as the site of the Δssa2 defect; Δssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Δssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Δssa2 Vid vesicles, providing Δssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.

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Stimulation of the Human Heat Shock Protein 70 Promoter in Vitro by Simian Virus 40 Large T Antigen

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)71601-3 ◽

1989 ◽

Vol 264 (27) ◽

pp. 16160-16164

Author(s):

I C Taylor ◽

W Solomon ◽

B M Weiner ◽

E Paucha ◽

M Bradley ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Large T Antigen ◽

Human Heat Shock Protein ◽

Stimulation Of

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Επικεκαλυμμένες ενδαγγειακές προθέσεις και επαναστένωση μετά από αγγειοπλαστική

10.12681/eadd/36988 ◽

2014 ◽

Author(s):

Δημήτριος Λυσίτσας

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

Low Dose ◽

High Dose ◽

Hsp 70

Εισαγωγή: Η υπερπλασία του έσω χιτώνα παίζει μείζων ρόλο στην επαναστένωση (in-stentrestenosis). Στην παρούσα μελέτη αξιολογήσαμε in vitro την επίδραση της D-24851(κυτταροτοξική ουσία που σταματά τον κυτταρικό κύκλο στο στάδιο G2-M) στονπολλαπλασιασμό των λείων μυϊκών κυττάρων και μελετήσαμε την ασφάλεια και τηνδραστικότητα μίας ενδαγγειακής πρόθεσης (stent) επικαλυμμένης με πολυμερή ουσία πουαπελευθερώνει την D-24851, στην αναστολή της υπερπλασίας του έσω χιτώνα χωρίς ναεμποδίζει την αναγεννητική ικανότητα του ενδοθηλίου σε in vivo πειραματικό μοντέλο.Υλικό και Μέθοδοι: Γυμνά μεταλλικά stent (n=6), stent επικαλυμμένα μόνο με πολυμερήουσία (polymer-coated, n=7) και stent επικαλυμμένα με πολυμερή ουσία πουαπελευθερώνουν 31±1μg (low-dose, n=7), 216±8 μg (high-dose, n=6) ή 1774±39 μg(extreme-dose, n=5) της D-24851 εμφυτεύτηκαν στις μηριαίες αρτηρίες λευκών New Zealandκουνελιών. Τα πειραματόζωα θυσιάστηκαν στις 28 ημέρες για ιστομορφομετρική ανάλυση.Για την αξιολόγηση της ενδοθηλιακής αναγέννησης στις 90 ημέρες, 12 πειραματόζωαχρησιμοποιήθηκαν για την τοποθέτηση polymer-coated (n=3), low dose (n=3), high dose(n=3) or extreme dose (n=3) ενδαγγειακών προθέσεων.Αποτελέσματα: In vitro η D-24851 αναστέλλει την υπερπλασία των λείων μυϊκών κυττάρωνκαι επάγει την απόπτωση τους χωρίς να αυξάνει την επαγωγή της heat shock protein 70(HSP-70), μία κυτταροπροστατευτική και αντι-αποπτωτική πρωτεΐνη. Η θεραπεία με lowdoseD-24851 stents συνδυάστηκε με 38% (P=0.029) μείωση της υπερπλαστικής περιοχήςτου έσω χιτώνα και 35% (P=0.003) μείωση της επι τοις εκατό στένωσης του αυλού σεσύγκριση με τα γυμνά μεταλλικά stents. Ο τραυματισμός και η φλεγμονή του αρτηριακού τοιχώματος δεν παρουσίασαν σημαντικές διαφορές μεταξύ των ομάδων. Τα επικαλυμμέναμόνο με πολυμερή ουσία stents εμφάνισαν παρόμοια ανάπτυξη νεοιστού σε σύγκριση με ταγυμνά μεταλλικά stents. Ωστόσο, όλες οι ομάδες των stents με D-24851 παρουσίασαν ατελήενδοθηλιοποίηση συγκρινόμενα με τα polymer-coated stents.Συμπεράσματα: Οι επικεκαλυμμένες ενδαγγειακές προσθέσεις με πολυμερή ουσία καιχαμηλη δόση D-24851 μειώνουν σημαντικά την υπερπλασιά του έσω χιτώνα. Λόγω τηςατελούς ενδοθηλιοποίησης, μακράς διάρκειας μελέτες είναι απαραίτητες για ναπιστοποιήσουν ότι η αναστολή του νεοιστού παραμένει και μετά τις 28 ημέρες.

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Heat shock protein 70 / MAGE-1 tumor vaccine can enhance the potency of MAGE-1–specific cellular immune responses in vivo

Cancer Immunology Immunotherapy ◽

10.1007/s00262-004-0536-6 ◽

2004 ◽

Vol 53 (9) ◽

pp. 825-834 ◽

Cited By ~ 13

Author(s):

Jing Ye ◽

Guang-Sheng Chen ◽

Hong-Ping Song ◽

Zeng-Shan Li ◽

Ya-Yu Huang ◽

...

Keyword(s):

Heat Shock ◽

Immune Responses ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

Tumor Vaccine ◽

Cellular Immune Responses ◽

Cellular Immune

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In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2069 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2069-2079 ◽

Cited By ~ 145

Author(s):

Ellen A. A. Nollen ◽

Jeanette F. Brunsting ◽

Han Roelofsen ◽

Lee A. Weber ◽

Harm H. Kampinga

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Heat Resistance ◽

Cell Line ◽

Heat Shock Protein 70 ◽

Chaperone Activity ◽

Luciferase Reporter ◽

Reporter Protein ◽

Regulated Gene Expression

ABSTRACT Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.

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In vivo analysis of heat-shock-protein-70 induction following pulsed laser irradiation in a transgenic reporter mouse

Journal of Biomedical Optics ◽

10.1117/1.2904665 ◽

2008 ◽

Vol 13 (3) ◽

pp. 030501 ◽

Cited By ~ 33

Author(s):

Caitlin E. O’Connell-Rodwell ◽

Mark A. Mackanos ◽

Dmitri Simanovskii ◽

Yu-An Cao ◽

Michael H. Bachmann ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Laser Irradiation ◽

Heat Shock Protein 70 ◽

Pulsed Laser ◽

Reporter Mouse ◽

In Vivo Analysis ◽

Pulsed Laser Irradiation

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Differential Neuroprotection from Human Heat Shock Protein 70 Overexpression in in Vitro and in Vivo Models of Ischemia and Ischemia-like Conditions

Experimental Neurology ◽

10.1006/exnr.2000.7614 ◽

2001 ◽

Vol 170 (1) ◽

pp. 129-139 ◽

Cited By ~ 103

Author(s):

Jong Eun Lee ◽

Midori A. Yenari ◽

Guo Hua Sun ◽

Lijun Xu ◽

Michelle R. Emond ◽

...

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

In Vivo Models ◽

Human Heat Shock Protein

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GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

Molecular Biology of the Cell ◽

10.1091/mbc.7.12.1921 ◽

1996 ◽

Vol 7 (12) ◽

pp. 1921-1937 ◽

Cited By ~ 119

Author(s):

R Murphy ◽

J L Watkins ◽

S R Wente

Keyword(s):

Saccharomyces Cerevisiae ◽

Schizosaccharomyces Pombe ◽

Nuclear Pore Complex ◽

Nuclear Export ◽

Protein Import ◽

Nuclear Transport ◽

Complex Structure ◽

Striking Similarity ◽

Nuclear Pore ◽

Pore Complexes

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

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