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

First published online 19 August 2008
doi: 10.1242/jcs.028696

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 Prosser, D. C. Articles by Ngsee, J. K. Search for Related Content PubMed PubMed Citation Articles by Prosser, D. C. Articles by Ngsee, J. K. Social Bookmarking                         What's this?

FFAT rescues VAPA-mediated inhibition of ER-to-Golgi transport and VAPB-mediated ER aggregation

Neuroscience, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Avenue, Ottawa ON, K1Y 4E9 Canada

View larger version (65K): [in this window] [in a new window]   Fig. 1. Subcellular localisation of WT and P56S VAPA and VAPB. CHO cells were transfected with either FLAG-tagged WT or P56S mutant in the absence of FFAT (top row) or with FFAT (bottom row). The proteins were visualised with anti-FLAG (red) and anti-Calreticulin (green). Scale bar: 10 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 2. Time course of VSVGts045-GFP trafficking in CHO cells transfected with WT or mutant VAPA and VAPB in the absence of FFAT. VSVGts045-GFP was trapped in the ER at 42°C for 6 hours and shifted to 32°C. The cells were fixed with 4% paraformaldehyde at the indicated time (minutes). Scale bar: 10 µm.

View larger version (111K): [in this window] [in a new window]   Fig. 3. Time course of VSVGts045-GFP trafficking in CHO cells transfected with WT or mutant VAPA and VAPB in the presence of FFAT. The conditions were as described in Fig. 2. Scale bar: 10 µm.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Effect of VAPA on transport of CgB-GFP(S65T). CHO cells were cotransfected with CgB-GFP(S65T) and control or VAPA (A) or VAPB (B) wild-type (WT) and mutant (P56S) vectors. The cells were incubated at 15°C for 2 hours and rapidly shifted to 37°C for 30 minutes before fixation and staining with anti-Calreticulin (Red). Scale bar: 10 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Effect of VAPA and VAPB on VSVG folding. VSVGts045-GFP was trapped in the ER at 42°C for 6 hours in transfected CHO cells and shifted to 32°C for 0 or 30 minutes before paraformaldehyde fixation. VSVG was detected by its GFP signal (top panel) or with the conformation-specific I14 antibody, which recognises properly folded VSVG (lower panel). Scale bar: 10 µm.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Effect of VAPA and VAPB on ER-vesicle budding. VSVGts045-Myc was trapped in the ER and ER-vesicle budding was reconstituted in vitro in perforated cells. Released VSVG-containing vesicles were collected by centrifugation and subjected to quantitative western immunoblot. The amount of VSVGts045 in vesicles released from semi-intact cells incubated at 4°C was subtracted from that in cells incubated at 32°C and the values were normalised as a percentage of the total cellular VSVG signal. Vehicle (DMSO) control (solid bars), FFAT (grey bars) and AAAT peptides (white bars) were added at 62.5 µM final concentration to the budding assay where indicated. The values represent mean ± s.e.m.

View larger version (33K): [in this window] [in a new window]   Fig. 7. FRAP analysis in the absence or presence of coexpressed FFAT motif. VSVGts045-GFP was trapped in the ER and shifted to 32°C immediately before a small area was photobleached. Fluorescence signal recovery was measured at 20 second intervals over a 10 minute period and values were normalised to the pre-bleach value. Cells were transfected with control vector (blue), VAPA-WT (red), VAPA-P56S (green), VAPB-WT (orange) or VAPB-P56S (purple) (A) or cotransfected with FFAT-expressing vector (B).

View larger version (39K): [in this window] [in a new window]   Fig. 8. Association of VAPA and VAPB with polymerised microtubules. (A) Detergent-solubilised extracts of transfected cells were pulled down with polymerised microtubules. The `input' panels show 25% of the input signal. A-WT, wild-type VAPA; A-N, N-terminal deletion VAPA; A-P56S, mutant P56S VAPA; B-WT, wild-type VAPB; B-N, N-terminal deletion VAPB. (B) Quantitative analysis of VAPA and VAPB recovered with polymerised microtubules in the presence of AAAT or FFAT peptide at the indicated concentrations. Values were normalised to the amount of VAPA or VAPB recovered in the absence of either peptide. Filled symbols represent microtubule association of VAPA-WT, VAPA-P56S and VAPB-WT in the presence of AAAT peptide whereas open symbols represent association of VAPA-WT, VAPA-P56S and VAPB-WT in the presence of FFAT peptide.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter