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First published online 27 July 2004
doi: 10.1242/jcs.01274

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Ribosome-translocon complex mediates calcium leakage from endoplasmic reticulum stores

Fabien Van Coppenolle^1,*,, Fabien Vanden Abeele^1,*, Christian Slomianny¹, Matthieu Flourakis¹, John Hesketh², Etienne Dewailly¹ and Natalia Prevarskaya¹

¹ Laboratoire de Physiologie Cellulaire, INSERM EMI 0228, Université de Lille 1, Bâtiment SN3, 59655 Villeneuve d'Ascq CEDEX, France
² School of Cellular and Molecular Biosciences, University of Newcastle, Agriculture Building, Kings Road, Newcastle-upon-Tyne, NE1 7RU, UK

View larger version (11K): [in a new window]   Fig. 1. Puromycin treatment reduces the thapsigargin-induced calcium release from the ER. (A) Traces represent the [Ca2+]c measured in 2 µM fura-2 AM loaded LNCaP cells under control conditions or after a 1-hour incubation with 200 µM puromycin or 1.8 mM cycloheximide. Application of 1 µM thapsigargin (TG) produced an increase in [Ca2+]c as a result of the inhibition of SERCA pumps. All measurements were made at room temperature in Ca2+-free HBSS. (B) Means of the peak values of thapsigargin responses under control conditions (n=34), with 1.8 mM cycloheximide (n=35) and with 200 µM puromycin (n=78).

View larger version (13K): [in a new window]   Fig. 2. Puromycin reduces the free Ca2+ concentration within the ER lumen. (A,B) Typical [Ca2+]ER traces from mag-fura-2 AM loaded LNCaP cells in response to 1 µM ionomycin (IM) (A) and 200 µM puromycin (B). The inset panels show individual fluorescence intensities at 340 nm (Ca2+-insensitive) and 380 nm (Ca2+-sensitive) excitation wavelengths. Note that application of ionomycin results in a drop in the mag-fura-2 ratio. (C) Cumulative data (mean±s.e.m.) for control and puromycin-treated LNCaP cells on the percentage of calcium release from internal stores.

View larger version (14K): [in a new window]   Fig. 3. Anisomycin blocks the calcium leak from intracellular stores induced by puromycin. (A,B) The intracellular stores of LNCaP cells were loaded with the calcium indicator mag-fluo-4. Recordings of the leak from LNCaP cells, permeabilized with digitonin in two representative LNCaP cells in response to the application of puromycin are shown. Addition of puromycin induced a slow reduction in [Ca2+]ER (A) whereas anisomycin pre-treatment inhibits passive Ca2+ leakage through the ER in response to puromycin application (B). (C) Cumulative data (mean±s.e.m.) for control and anisomycin-treated LNCaP cells of percentage of calcium released from internal stores.

View larger version (19K): [in a new window]   Fig. 4. Calcium leak from the intracellular stores induced by puromycin occurs independently of IP3 and RyR stores. (A) Time course of a typical experiment of the passive Ca2+ leak induced by puromycin in digitonin-permeabilized LNCaP cells treated with ryanodine and heparin. At the end of experiment, ionomycin was added to indicate the size of the total releasable pool. (B) Sequential application of puromycin and IP3/cADPr to indicate the size of each releasable pool. At the end of experiment, ionomycin was also added to indicate the size of the total releasable internal calcium store. (C) Cumulative data (mean±s.e.m.) for the percentage of calcium released from internal stores.

View larger version (101K): [in a new window]   Fig. 5. Colocalization of the 60S ribosomal subunit with translocon protein Sec 61. (A-C) Sec 61, recognized by polyclonal Sec 61 antibodies, was detected using FITC-labeled secondary antibodies. 60S ribosomal subunit, recognized by polyclonal antibodies, was detected using Texas-Red-labeled secondary antibodies. No staining was seen in the absence of primary antibodies (data not shown). Cells were analyzed using a Zeiss LSM 510 confocal laser scanning system connected to a Zeiss Axiovert 200 M. Images were collected separately for each channel (Texas-Red at 563 nm and FITC at 488 nm excitation) and merged as indicated. The pictures were taken after a 1-hour treatment under control conditions (A), with 200 µM puromycin (B) or with 1.8 mM cycloheximide (C). Bar, 20 µm. (D) Correlation coefficients calculated according to reported methods (Manders et al., 1993). Puromycin slightly reduced colocalization of the Sec61 and ribosome labels. (E-G) Transmitted electron micrographs of LNCaP cells in control conditions (E), or after a 1-hour treatment with 200 µM puromycin (F) or 1.8 mM cycloheximide (G). Bar, 200 nm.