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First published online 29 August 2006
doi: 10.1242/jcs.03172

Journal of Cell Science 119, 3888-3900 (2006)
Published by The Company of Biologists 2006
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Starvation and ULK1-dependent cycling of mammalian Atg9 between the TGN and endosomes

Andrew R. J. Young1, Edmond Y. W. Chan1, Xiao Wen Hu1, Robert Köchl1, Samuel G. Crawshaw2, Stephen High2, Dale W. Hailey3, Jennifer Lippincott-Schwartz3 and Sharon A. Tooze1,*

1 Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK
2 Faculty of Life Sciences, University of Manchester, Smith Building, Oxford Road, Manchester, M13 9PT, UK
3 NIH / NICHD / CBMB, Bldg 18T Room 101, 9000 Rockville Pike, Bethesda, MD, 20892, USA


Figure 1
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  		Fig. 1. Distribution and topology of mAtg9. (A) Proposed topology of mAtg9 shown with seven transmembrane domains (TMDs). The conserved Atg9 PFAM domain is shown in green. Black dots represent potential N-glycosylation sites. Antibodies raised against the N- and C-termini were designated STO215 and STO219, respectively. (B) Immunofluorescence in HEK293 cells with Alexa Fluor-488- and Alexa Fluor-555-conjugated mAtg9 antibodies. (C) Indirect immunofluorescence of HA-tagged mAtg9 and anti-Atg9 (STO219). Bar, 10 µm. (D) HEK293 PNS was treated with glycosidases, EndoH (lane 1) or PNGaseF (lane 3). UN, untreated (lane 2). PNGaseF treatment altered the migration of mAtg9 from 105 kDa to 75 kDa. (E) Lysates from HEK293 cells transiently transfected with wt HA-mAtg9 (lanes 1 and 2), HA-mAtg9 N99D (lanes 3 and 4), N224/507D (lanes 5 and 6) or N99/224/507D (lanes 7 and 8) were treated (+) or not (-) with PNGaseF. The N99D and N99/224/507D mutants were not glycosylated (lower band, *), whereas the N224/507D mutant was glycosylated (upper band, arrowhead) and sensitive to PNGaseF. (F) An in vitro synthesized 183 aa N-terminal mAtg9 fragment was treated (+) or not (-) with EndoH. The wt sequence fragment (lanes 1 and 2) was glycosylated (upper band, arrowhead), and sensitive to EndoH, whereas the N99D mutant fragment (lanes 3 and 4) was not (lower band, *). (G) Topology of the N-terminus: ER microsomes, containing the 32 kDa, 183 aa fragment (lane 1), were treated with proteinase K (Prot K) in the absence of TX-100 (lane 2) resulting in two bands migrating at ~14 kDa and ~18 kDa (*), which correspond to the protected glycosylated and non-glycosylated forms of the TM1, TM2 and their inter-TMD loop (see Model A). No bands were detected after Prot K treatment in the presence of Triton X-100 (TX-100, lane 3). The 183 aa fragment could be immunoprecipitated using anti-N-terminal STO215 (lane 4), but not after Prot K treatment in the absence (lane 5) or presence (lane 6) of detergent, consistent with the removal of the N-terminal epitope (Model A). Note: If the N-terminus were lumenal (see Model B), then a ~25 kDa band would have been detected. The numbers shown on the models refer to amino acid position. C, control, PI, pre-immune serum, Tot, totals, i.e. non-IPs, Cyt, cytosol, Lum, lumen. (H) Topology of C-terminus. Full-length in-vitro-synthesized mAtg9 was immunoprecipitated using anti-C-terminal STO219 (lane 1, arrowhead). After Prot K treatment of the microsomes in the absence of TX-100 no fragment was immunoprecipitated (lane 2), consistent with removal of the C-terminal epitope (Model A). Note: If the C-terminus were lumenal, then a ~40 kDa fragment would have been expected (Model B). Controls are as for panel G.

 

Figure 2
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  		Fig. 2. In vivo topology analysis of mAtg9. Plasmids YFP-HLA-A2-CFP and mRFPmAtg9 were transiently transfected into NRK cells. YFP on the N-terminus is in the ER lumen and CFP on its C-terminus is cytosolic. (A) Pre-treatment images. (B) Images captured after 2 minutes of permeabilization with digitonin. (C) Images captured 5 minutes after subsequent trypsin addition. At the concentration used, digitonin permeabilized the plasma membrane but not the ER. Lumenal YFP on the N-terminus of HLA-A2 was protected from the trypsin, but the C-terminal CFP and the mRFP on the N-terminus of mAtg9 were degraded by the trypsin and are therefore cytosolic. All images were captured using identical imaging parameters.

 

Figure 3
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  		Fig. 3. mAtg9 localizes to the trans-Golgi network. HEK293 cells were labelled with antibodies to mAtg9 and GM130 (A,B) or TGN46 (C,D). Cells in B and D were treated for 30 minutes with nocodazole (noc). Insets illustrate the colocalization after noc treatment. Bar, 10 µm. (E) Immunogold labelling of mAtg9 (10-nm gold, arrows) and GM130 (5-nm gold, arrowheads (left panel) in HEK293 cells. G, Golgi. Bars, 200 nm.

 

Figure 4
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  		Fig. 4. mAtg9 is present on late endosomes. (A) Western blots of fractions from an endosome preparation gradient from rat liver using antibodies for EEA1 (early endosomes), mAtg9, CI-MPR, TGN38 (TGN), p58 and PDI (ER). To label late endosomes, HRP-biotin was internalized by perfusion for 10 minutes followed by a 20-minute chase before homogenization. HRP-biotin was detected using ExtraAvidin-HRP. Signals were quantified by densitometry, performed using ImageJ. Data are representative of two independent experiments. (B) mAtg9 colocalizes with the CI-MPR in indirect immunofluorescence on HEK293 cells. Inset is enlargement of the peripheral staining in the merge. Bar, 10 µm. (C) Cryoimmunogold labelling of mAtg9 (red arrowheads, 10-nm gold), CI-MPR (green arrowheads, 15-nm gold), on HEK293 cells labelled with 6-nm conjugated BSA-Gold (arrows) internalized for 2 hours. Bar, 200 nm.

 

Figure 5
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  		Fig. 5. mAtg9 colocalizes with Rab6 on Golgi membranes, and Rab7 and 9 on late endosomes. HEK293 cells were transiently transfected with GFP-Rab6 (A), GFP-Rab7 (B) and GFP-Rab9 (D), then 24 hours later fixed and labelled with the mAtg9 antibody. Cells were also labelled with antibodies to endogenous Rab7 (C) and 9 (E), the inset is an enlargement of the merge to show the peripheral staining. For Rab7 staining, cells were extracted using 0.05% (w/v) saponin before fixation. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. mAtg9 and autophagy. (A) HEK293/GFP-LC3 cells starved for 2 hours were labelled with antibodies for both endogenous Rab7 and mAtg9. White arrowhead points to GFP-LC3, mAtg9, Rab7 colocalization, blue to GFP-LC3 and mAtg9, and yellow to GFP-LC3 and Rab7. Bar, 10 µm. (B) HEK293/GFP-LC3 were treated with the siRNA for 72 hours, starved for 2 hours with ES medium or not (culture in FM), and extracted. Conversion of GFP-LC3-I to GFP-LC3-II was assessed using a GFP antibody. (C) Long-lived protein degradation was assessed 72 hours after siRNA addition in triplicate, as described in Materials and Methods. *P<0.01.

 

Figure 7
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  		Fig. 7. mAtg9 and CI-MPR redistribute upon starvation in a reversible, PtdIns 3-kinase-dependent manner. HEK293 cells were labelled with antibodies against mAtg9 and CI-MPR (A-D) or TGN46 (E-G). Cells had first been treated with either rapamycin (C) for 2 hours, or for 2 hours in ES medium in the presence (D,F) or absence (B,E) of 65 µM LY294002. (G) Cells had been starved for 2 hours in ES medium, before incubation in FM for 2 hours in the presence of 10 µg/ml cyclohexamide. Note: cyclohexamide was also included in the final 15 minutes in ES medium). Bar, 10 µm.

 

Figure 8
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  		Fig. 8. The starvation-induced dispersal of mAtg9 can be blocked by knockdown of ULK1 but not ULK2. HEK293 cells were treated with either control siRNA (A,B,E,F) or siRNA to either ULK1 (C,G) or ULK2 (D) and incubated in FM (A,E) or for 2 hours in ES medium (B-D,F,G). The cells were then fixed and labelled with antibodies against mAtg9 and TGN46 or CI-MPR. The starvation-induced dispersal of both mAtg9 (B,F) and CI-MPR (F) could be blocked by siRNA-mediated knockdown of ULK1 (C,G) but not ULK2 (D). Bars, 5 µm.

 

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© The Company of Biologists Ltd 2006