Accumulation of undegraded autophagosomes by expression of dominant-negative STX17 (syntaxin 17) mutants

<p>Macroautophagy/autophagy, which is one of the main degradation systems in the cell, is mediated by a specialized organelle, the autophagosome. Purification of autophagosomes before fusion with lysosomes is important for both mechanistic and physiological studies of the autophagosome. Here, we report a simple method to accumulate undigested autophagosomes. Overexpression of the autophagosomal Qa-SNARE STX17 (syntaxin 17) lacking the N-terminal domain (NTD) or N-terminally tagged GFP-STX17 causes accumulation of autophagosomes. A HeLa cell line, which expresses GFP-STX17ΔNTD or full-length GFP-STX17 under the control of the tetracycline-responsive promoter, accumulates a large number of undigested autophagosomes devoid of lysosomal markers or early autophagy factors upon treatment with doxycycline. Using this inducible cell line, nascent autophagosomes can be easily purified by OptiPrep density-gradient centrifugation and immunoprecipitation. This novel method should be useful for further characterization of nascent autophagosomes.</p

Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite <em>Plasmodium falciparum</em>

<div><p>Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite <em>Plasmodium falciparum</em> (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although <em>Plasmodium</em> parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.</p> </div

PfAtg8 is associated with membranes.

<p>(A) Specificity of the two independently generated anti-PfAtg8 antibodies (#1 and #2). Crude antisera and purified antibodies were used for immunoblotting of lysates of asynchronized <i>P. falciparum</i> parasites. (B) Expression of PfAtg8 increases during the erythrocytic stage of development. Highly synchronized <i>P. falciparum</i> parasites were collected at 0, 12, 24, 32, and 40 h after invasion. The duration of one cycle of the erythrocyte stage was approximately 42 h. Expression levels of PfAtg8 were analyzed by immunoblotting. An antibody against HSP70 was used as a loading control. (C) PfAtg8 exogenously expressed in mammalian cells (lane 1), endogenous PfAtg8 expressed in <i>P. falciparum</i> (lane 2), and the mixture of these two samples were subjected to immunoblot analysis using anti-PfAtg8 antibody. (D) Lysates of asynchronized <i>Plasmodium</i> were separated into low-speed (13,000×<i>g</i>) pellet (LSP), high-speed (100,000×<i>g</i>) pellet (HSP), and high-speed supernatant (HSS) fractions, and analyzed by immunoblotting using anti-PfAtg8 antibody. (E) The LSP fraction prepared in (D) was treated with 2 M urea or 2% Triton-X 100 and separated into 100,000×<i>g</i> pellet (P) and supernatant (S). (F) Infected erythrocytes were cultured in the presence of the indicated concentration of chloroquine and expression of PfAtg8 was analyzed.</p

PfAtg8 is associated with the apicoplast membrane.

<p>(A) <i>P. falciparum</i> FCR3 parasites at the schizont stage were analyzed by immunoelectron microscopy (immunogold and silver enhancement method) with an antibody against PfAtg8 (#1). (a) A schizont in an erythrocyte. (b) A magnified image of the area indicated in (a). (c) Another typical image of a PfAtg8-positive structure. (B) <i>P. falciparum</i> transfectant expressing ACP-GFP was analyzed as in panel (A) with an antibody against GFP. A, apicoplast; Mt, mitochondrion. Scale bars, (A, a) 1 μm, (A, b and c, and B) 200 nm.</p

(A) NIH3T3 cells stably expressing GFP-FIP200 were cultured in complete or starvation medium for 120 min and the GFP signal was observed

(B) NIH3T3 cells stably expressing GFP-FIP200 were cultured in starvation medium for 120 min and then subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 568–conjugated secondary antibody. Bars, 20 μm. More than 90% of GFP-FIP200 dots were positive for Atg16L1. (C) NIH3T3 cells stably expressing GFP-ULK1 were starved for 120 min and then subjected to immunofluorescence microscopy using anti-FIP200 antibody and Alexa Fluor 568–conjugated secondary antibody. Black squares indicate the enlarged areas shown in insets. Bar, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

PfAtg8 localizes to the apicoplast.

<p><i>P. falciparum</i> FCR3 (A–E) and <i>P. falciparum</i> 3D7 transfected with ACP-GFP (F–H) were stained with the indicated organelle markers and visualized by confocal microscopy (because ACP-GFP was not uniformly expressed, some merozoites displayed only faint GFP signals). Anti-PfAtg8 antibody #1 was used in (A–F), and anti-PfAtg8 antibody #2 was used in (G). Apical membrane antigen 1 (AMA1) as a microneme marker (A), rhoptry-associated protein 1 (RAP1) as a rhoptry body marker (B), rhoptry neck protein 2 (RON2) as a rhoptry neck marker (C), the ring-infected erythrocyte surface antigen (RESA) as a dense granule marker (D), MitoTrackerRed CMXRos as a mitochondria marker (E), ACP-GFP (F–H) and the organellar histone-like protein PfHU (H) as an apicoplast marker were used. Scale bar, 1 μm.</p

(A and B) NIH3T3 cells stably expressing GFP-ULK1 (A) and -ULK2 (B) were cultured in complete or starvation medium for 30 min

They were then cultured in fresh complete medium for an additional 30 min (starvation→complete). (C and D) NIH3T3 cells stably expressing GFP-ULK1 (C) and -ULK2 (D) were cultured in starvation medium for 120 min. The cells were fixed, permeabilized, and subjected to immunofluorescence microscopy using anti-Atg16L1 antibody and Alexa Fluor 660–conjugated secondary antibody. More than 90% of GFP-ULKs dots were positive for Atg16L1. (E–H) Wild-type (E and G) and (F and H) MEFs were transfected with retroviral vectors encoding GFP-ULK1 and -ULK2. MEFs stably expressing GFP-ULK1 (E and F) and -ULK2 (G and H) were cultured in complete or starvation medium for 120 min. The cells were fixed and examined by fluorescence microscopy. Bars, 20 μm.<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

(A) and MEFs stably expressing GFP-ULK1 or -ULK2 were cultured in starvation medium for 120 min

The cells were fixed and examined by fluorescence microscopy. Bar, 20 μm. (B) and MEFs were cultured in complete medium. The cell lysates were subjected to immunoblot analysis with antibodies against ULK1 or HSP90 (loading control). (C) Phosphatase sensitivity of ULK1. and MEFs were cultured in complete medium. ULK1 was immunoprecipitated from cell lysates and treated with λ phosphatase for 30 min at 30°C in the absence or presence of phosphatase inhibitors (1 mM NaVO, 50 mM KF, 15 mM NaPO, and 1 mM EGTA).<p><b>Copyright information:</b></p><p>Taken from "FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells"</p><p></p><p>The Journal of Cell Biology 2008;181(3):497-510.</p><p>Published online 5 May 2008</p><p>PMCID:PMC2364687.</p><p></p

PfAtg8 localization is not affected by chloroquine or wortmannin treatment.

<p><i>P. falciparum</i> transfectant expressing ACP-GFP was treated with chloroquine (100 or 300 nM) (A), or wortmannin (10 μM) (B) for 2 h. Scale bar, 1 μm.</p

PfAtg8 localizes to tubular and branched apicoplasts.

<p><i>P. falciparum</i> transfectant expressing ACP-GFP at late trophozoite and early schizont stages was stained with anti-GFP and anti-PfAtg8 antibodies and MitoTrackerRed CMXRos, and visualized by confocal microscopy. Scale bar, 1 μm.</p