QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00802

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Emery, G. Articles by Gruenberg, J. Search for Related Content PubMed PubMed Citation Articles by Emery, G. Articles by Gruenberg, J.

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics

Gregory Emery^1,*, Robert G. Parton², Manuel Rojo^1, and Jean Gruenberg^1,

¹ Department of Biochemistry, University of Geneva, 30 quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland
² Institute for Molecular Bioscience, Centre for Microscopy & Microanalysis, and School of Biomedical Sciences, University of Queensland, 4072 Brisbane, Australia

View larger version (26K): [in a new window]   Fig. 1. Outline of p24 proteins and constructs. (A) Alignment of the cytoplasmic tails of p24 proteins and mutants (except chimeras) used in this study. White box: double-phenylalanine motif potentially involved in COP-II binding; gray box: positively charged amino acids, which are proposed to be involved in COP-I binding and retention in the early biosynthetic pathway. We termed p28 a novel putative member of the p24 family identified as a human gene evolutionarily conserved in Caenorhabditis elegans by comparative proteomics (accession number: Q9Y3A6). Tp24 was originally named putative T1/ST2 receptor binding protein (see Emery et al., 2000). (B) Schematic representation of p23 and p25 chimera used in this study. LD, lumenal domain; TM, transmembrane domain; CT, cytoplasmic tail. Domains of p23 are in light gray, CD4 in dark gray, and p25 in white.

View larger version (70K): [in a new window]   Fig. 2. p25SS transport to post-Golgi membranes. (A) HeLa cells were transfected with myc-p23SS, HA-p24SS or myc-p25SS, as indicated. Then, cells were either permeabilized and the distribution of the indicated proteins was revealed by immunofluorescence using antibodies against myc or HA, or not permeabilized (4°C) and the corresponding antibodies were added to the living cells at 4°C prior to fixation. (B) Cells transfected as in A were incubated for 16 hours at 37°C with antibodies against myc or HA to detect the indicated proteins, and then the distribution of the endocytosed antibodies was revealed by immunofluorescence. (C) Cells were triply transfected with untagged p23, HA-p24 and myc-p25SS. Then, cells were incubated for 16 hours at 37°C with antibodies as in B except that p23 was labeled with anti-lumenal domain antibodies. The distribution of the endocytosed antibodies was analyzed as in B. Scale bar: 10 µm.

View larger version (63K): [in a new window]   Fig. 3. Localization of early biosynthetic proteins. HeLa cells were transfected with mycp25SS alone and then incubated with anti-myc antibodies as in Fig. 2B, or triply cotransfected with p25SS, p23 and HA-p24 and then incubated with anti-HA antibodies. The distribution of antibody-tagged myc-p25SS or HA-p24 was then compared with those of the indicated proteins by indirect immunofluorescence. Cells transfected only with mycp25SS are shown in the panels that also illustrate the distribution of calnexin, ERGIC53, GRASP55 and p115, while triply co-transfected cells are shown in the panels that also illustrate the distribution of giantin and COP-I. The central column shows merged micrographs, color-coded as indicated. Scale bars: 10 µm.

View larger version (91K): [in a new window]   Fig. 4. Distribution of p25SS in the endocytic pathway. Cells were transfected with myc-p25SS and then incubated with rabbit anti-myc antibodies, as in Fig. 2B. Where indicated, rhodamine-labeled dextran was added to the incubation medium during the last 60 minutes of incubation. To label late endosomes, 5 µg/ml monoclonal anti-LBPA antibody (6C4) were co-endocytosed with anti-myc antibodies, to limit accessibility problems within late endosomes (a non-relevant control antibody does not accumulate intracellularly [not shown and (Kobayashi et al., 1998)]. At this low dose, 6C4 does not interfere with trafficking. Then, endocytosed antibodies were revealed as in Fig. 2B using appropriate secondary antibodies. The distribution of antibody-tagged p25SS was compared with that of endocytosed dextran or anti-LBPA antibodies, or to Lamp1 (using anti-Lamp1 antibodies), as indicated. The bottom row shows the distribution of endocytosed dextran and LBPA in untransfected control cells; the right column shows the merged images. Scale bars: 10 µm.

View larger version (76K): [in a new window]   Fig. 5. Clusters in endosomes cause a cholesterol storage defect. (A) Cells were transfected with myc-p25SS, incubated with anti-myc antibodies, as in Fig. 2B, and processed for electron microscopy to reveal the distribution of antibody-tagged p25SS clusters (arrows). The membrane of typical late endosomes is outlined by arrowheads. (B) HeLa cells were transfected with myc-p25SS and then treated with 0.5 µg/ml ASSP mutant of aerolysin for 16 hours at 37°C. The distribution of antibody-tagged p25SS was then compared to that of Lamp1, as in Fig. 4. The toxin mutant causes late endosome vacuolation. (C) Cells were transfected with myc-p25SS or myc-p23-TMCD4 and processed for indirect immunofluorescence using anti-myc antibodies, and filipin to reveal cholesterol distribution. The arrow points to cholesterol accumulation in a p25SS-transfected cell. Scale bars: 10 µm.

View larger version (71K): [in a new window]   Fig. 6. Cholesterol accumulation and motility inhibition. (A) Cells were transfected with mycp25SS, incubated with anti-myc antibodies, as in Fig. 2B, and then processed for triple-channel immunofluorescence. The distribution of antibody-tagged p25SS (red) was compared to that of Lamp1 (green) and cholesterol (blue, directly revealed with filipin) in a merged image. A high magnification view of the outlined area is shown in color for each compound, emphasizing the differences in the distribution of p25SS, Lamp1 and cholesterol. (B) Cells were transfected with CD63-GFP alone or together with myc-p25SS. Transfected cells were identified with anti-myc antibodies added to the living cells (not shown). The motility of late endosomes containing CD63-GFP was analyzed (Lebrand et al., 2002) by collecting images (200 mseconds exposure time) every second over a 25-second period. Then, all images were stacked. When represented in this manner, a moving object shifts position, thus creating a series of overlapping or closely associated spots that reveals its track (left panels), as in the control cells. Initial and final positions were color-coded after electronic conversion of the first and last pictures in the sequence to red and green, respectively, so that a moving object appears both red and green, and an immobile object yellow (middle panels). Examples of the traces of individual elements are shown, and highlighted by boxes in the right panels. (C) In cells co-transfected with CD63GFP and p25SS, as in B, the direct distances between initial and final positions of CD63GFP-labeled vesicles were quantified after 25 seconds (and not the actual trajectory followed by vesicles), as indicated (700 vesicles were analyzed and standard deviations are shown). Scale bars: (A,B) 10 µm.

View larger version (116K): [in a new window]   Fig. 7. Cell surface clusters. (A) Cells were transfected with myc-p25SS, incubated without permeabilization at 4°C with the ASSP mutant of aerolysin. Then, cells were treated simultaneously with chicken anti-aerolysin antibodies, Cy3-anti-myc antibodies (color-coded in green) and Cy5-transferrin (color-coded in red), to avoid possible accessibility problems due to raft clustering. Finally, raft domains were further clustered and visualized using anti-chicken secondary antibodies (color-coded in blue). An analysis by triple-channel fluorescence shows the distribution of each marker in a transfected cell, and the inset shows a high magnification view of the outlined area. (B) As in A, except that cells were transfected with p23-CD4TM, and transferrin was omitted. A high magnification view of the plasma membrane shows the distribution of ASSP, p23-CD4TM and the merged image (left). (C) Cells transfected with p25SS were processed for electron microscopy. The micrograph shows a high magnification view of the plasma membrane with immunogold labeling of p25SS. Scale bar: 2 µm.