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First published online 20 June 2006
doi: 10.1242/jcs.03047

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Atg9 sorting from mitochondria is impaired in early secretion and VFT-complex mutants in Saccharomyces cerevisiae

¹ Department of Cell Biology, Cell Microscopy Center and Institute of Biomembranes, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
² Life Sciences Institute and Departments of Molecular, Cellular and Developmental Biology and Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

View larger version (48K): [in a new window]   Fig. 1. Early sec mutants block cycling of Atg9. The structure of the peripheral ER and the mitochondrial reticulum is altered in the sec12 mutant at nonpermissive temperatures. (A,B) The wild-type (SEY6210) and sec12 (FBY217) strains both transformed with the plasmid expressing Spo7-GFP (YEplac122-TRP1-SPO7-GFP) were grown at 24°C (A) and then transferred to 37°C (B) for 2 hours. Cells were labeled with MitoFluor Red and viewed before and after the temperature shift. Images of the fluorescent signal were acquired while focusing on either the center or the periphery of the cells. DIC, differential-interference-contrast images.

View larger version (58K): [in a new window]   Fig. 2. Atg9 sorting from mitochondria is impaired in sec12 and sed5 cells. (A,B,C) The sec12 atg1 Atg9-YFP (FRY237) or sed5 atg1 (FRY303) strains transformed with the vector carrying the atg1ts allele were grown at permissive temperature (24°C) (A; C, left panels) and then shifted to 37°C (B; C, right panels) for 1 hour. Cells were photographed before and after the temperature change; the cells in C were first subjected to mild fixation. (A,B) To visualize mitochondria, MitoFluor Red was added to the culture medium 30 minutes before taking the pictures. The YFP signal is green to facilitate comparison with MitoFluor Red. Control strains were atg1 Atg9-YFP (FRY138) carrying the plasmid expressing Atg1ts and sec12 Atg9-YFP (FRY154). Arrows indicate colocalization of Atg9-YFP and MitoFluor Red. Arrowheads indicate regions where Atg9-YFP is in close proximity to MitoFluor Red. DIC, differential-interference-contrast images.

View larger version (74K): [in a new window]   Fig. 3. Organization of actin cables is not affected in sec12 cells. The wild-type (SEY6210) and sec12 (FBY217) strains grown at 24°C were fixed, permeabilized and incubated with Texas Red-phalloidin (TR-phalloidin) as described in Materials and Methods before collecting fluorescence images. The sec12 mutant was also treated and imaged as above after being incubated at restrictive temperature for 90 minutes. DIC, differential-interference-contrast images.

View larger version (53K): [in a new window]   Fig. 4. Atg9 sorting from mitochondria is impaired in the absence of the VFT complex. The wild-type (SEY6210) and vps52 (PSY113) strains both transformed with the plasmid expressing Spo7-GFP (YEplac122-TRP1-SPO7-GFP) were grown at 30°C to an early log-phase in rich medium (A) or starved of nitrogen for 2 hours (B). Cells were labeled with MitoFluor Red and imaged as described in Fig. 1. The mitochondrial reticulum was disrupted in the absence of VPS52 in rich medium. DIC, differential-interference-contrast images.

View larger version (81K): [in a new window]   Fig. 5. Alteration of subcellular structures in the vps52 mutant. (A) Mitochondrial morphology is severely compromised in the absence of Vps52. The vps52 (PSY113) strain was grown in rich medium to an early log-phase and mitochondria labeled by adding MitoFluor Red prior to imaging. (B) Actin cables are aberrantly organized in the vps52 mutant. Cells grown as in panel A were fixed, permeabilized and incubated with Texas Red-phalloidin (TR-phalloidin) as described in Materials and Methods before collecting fluorescence images. DIC, differential-interference-contrast images.

View larger version (84K): [in a new window]   Fig. 6. Atg9 sorting from mitochondria is blocked in the vps52 strain in rich medium but not under starvation conditions. (A,B) The vps52 Atg9-YFP (FRY238) and vps52 atg1 Atg9-YFP (FRY192) strains were grown in rich medium (A) or starved in SD-N for 3 hours (B) and then photographed. Mitochondria were labeled by adding MitoFluor Red 30 minutes prior to imaging. Arrows indicate sites of colocalization between Atg9-YFP and MitoFluor Red. Arrowheads indicate regions where Atg9-YFP is in close proximity to MitoFluor Red. DIC, differential-interference-contrast images.

View larger version (48K): [in a new window]   Fig. 7. Atg9 trafficking from mitochondria is not affected in the absence of Vps4. The vps4 Atg9-YFP (FRY304) and vps4 atg1 Atg9-YFP (FRY305) strains were grown in rich medium, subjected to mild fixation and then photographed. Vacuoles were labeled by adding FM 4-64 FX 1 hour prior to imaging. The YFP signal is in green to facilitate comparison with FM 4-64 FX. Arrows indicate abnormally enlarged late endosomes typical of the vps4 mutant.

View larger version (16K): [in a new window]   Fig. 8. Model of subcellular Atg9 trafficking in S. cerevisiae. (A) Atg9 cycles between the PAS and mitochondria. Atg9 sorting from mitochondria is blocked by disruption of the ER-mitochondrial network by early sec mutations, deletion mutants that lack VFT-complex subunit and actin filament disruption. Once the double-membrane vesicle is completed and the function of Atg9 is no longer needed, the Atg1-Atg13 complex triggers the retrieval transport of this protein from the PAS (Reggiori et al., 2004a). Atg2, Atg18 and the PtdIns(3)P generated by the Atg14-containing PtdIns 3-kinase complex I also participate in this recycling event. (B) Inhibition of the Atg9 sorting event from mitochondria. The proper organization of the mitochondrial reticulum is maintained by both the cytoskeleton and a direct connection with the ER. Alteration of one of these structures provokes the disruption of the mitochondrial network, which blocks Atg9 trafficking. LatA treatment and deletion of each one of the subunits of the VFT complex affects actin filaments, whereas early sec mutations disrupt the ER peripheral network. In addition, actin also plays a direct role in Atg9 delivery and cargo recruitment to the PAS (Reggiori et al., 2005a).