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Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface

Marie Curie Research Institute, The Chart, Oxted, Surrey, RH8 0TL, UK

View larger version (25K): [in a new window]   Fig. 1. (a) Schematic representation of p32, including various predicted motifs. (b) Summary of p32 constructs p32-myc, flag-p32 and flag-p32-myc. The boxes indicate the epitope tags. (c) Typical staining pattern of the endogenous p32 in COS-1 cells observed with an anti-p32 polyclonal antibody.

View larger version (51K): [in a new window]   Fig. 2. N-terminal epitope tagging of p32 changes its distribution. Indirect immunofluorescence of COS-1 cells transfected with C-terminal-tagged p32-myc (a,b), double-tagged flag-p32-myc (c,d) and N-terminal-tagged flag-p32. (e,f). Cells were fixed 40 hours after transfection and incubated with antibodies against the c-myc epitope (a,c,e) or against the Flag epitope. Cells were analysed by confocal microscopy and typical images illustrated.

View larger version (57K): [in a new window]   Fig. 3. P32-myc localisation in mitochondria is blocked by N-terminal residues. Cells were transfected with p32-myc (panel a) and flag-p32-myc (panel b) and stained with anti-prohibitin (red) and anti-myc antibodies (green). The individual images are shown merged in the right-hand panel. Arrows indicate cells transfected expressing the p32 species. p32-myc and prohibitin showed almost complete co-localisation, appearing yellow in the merged image, whereas the flag-p32 appears in a distinct globular pattern showing little overlap with the mitochondria.

View larger version (72K): [in a new window]   Fig. 4. Localisation of flag-p32 compared to a number of ER-Golgi components. Staining with antibodies against markers for endogenous calreticulin, calnexin, -adaptin, and ß-COP are in red. Flag-p32 is shown in green. In each of the panels showing individual cellular components, cells expressing the p32 species are indicated by long arrows, whereas for comparison cells not expressing the p32 are shown by short arrowheads. Two separate fields (panels b,c) are illustrated for calreticulin relationship to flag-p32, whereas panel a shows the calreticulin pattern for the normally localised p32-myc. The individual images are shown merged in the right-hand panel.

View larger version (25K): [in a new window]   Fig. 5. Cell-surface expression of flag-p32 species. (a) Staining of non-permeabilised cells demonstrates cell-surface expression of flag-p32 constructs. COS-1 cells transfected with p32-myc or flag-p32-myc were fixed in 4% paraformaldehyde and analysed with anti-myc antibody. (b,c) Cell-surface biotinylation of p32 expression constructs. COS-1 cells were transfected with flag-p32 (lanes 1), flag-p32-myc (lanes 2) and p32-myc (lanes 3) and 40 hours later labelled with membrane-impermeable Sulfo-NHS-LC-biotin. (b) Total extracts (5x104 cells per lane). (c) Streptavidin-bound, cell-surface expressed, extract (5x105 cells per lane). Proteins were detected by western blot using the indicated antibodies. * indicates proteolytically cleaved p32.