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1.
FIG. 2

FIG. 2. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

In starved aut5Δ cells autophagic bodies accumulate in the vacuole. The cells were grown in yeast extract-peptone-dextrose and starved for nitrogen for 4 to 5 h in 1% potassium acetate medium at 30°C. The cells were then checked by light microscopy with Nomarski optics (bar in left panel, 20 μm; insets are enlarged twofold) or prepared for electron microscopy by permanganate fixation and epon embedding (bar in right panel, 1 μm). N, nucleus; V, vacuole.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
2.
FIG. 8

FIG. 8. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

The phosphatidylinositol 3-phosphate 5-kinase Fab1p (A) and Vps class E proteins (B to D) are required for targeting of Aut5-Ha. Cells expressing Aut5-Ha from a 2μm plasmid were grown at 25°C to log phase, fixed with formaldehyde, and spheroplasted. After treatment with antibodies to Ha and a secondary Cy3 antibody, the cells were analyzed by indirect immunofluorescence microscopy. From left to right, Nomarski optics (Nom), immunofluorescence microscopy (Aut5-Ha), and nuclear staining with DAPI (DNA/DAPI) are shown. In fab1Δ cells (A) Aut5-Ha is localized to the vacuolar membrane and the ER. In vps class E cells (B to D), Aut5-Ha is seen at the prevacuolar compartment (arrowheads) and to some extent at the vacuolar membrane and the ER. Bar, 10 μm.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
3.
FIG. 1

FIG. 1. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

Identification and characteristics of AUT5. (A) The AUT5 genetic locus and the isolated complementing genomic fragment are shown. (B) Aut5p shares homologies with AL033391 from C. albicans, SPAC23C4 from S. pombe, and pSI-7 from Cladosporium fulvum. Aut5p contains a putative lipase active-site motif from amino acids 326 to 335 (filled rectangle). Box filled with ×'s, potential transmembrane domain; hatched boxes, potential glycosylation sites. Alignment was created using CLUSTAL, and identical residues are shaded. (C) Hydrophobicity blot of Aut5p generated by using the method of Engelman et al. (10). One potential transmembrane region is predicted between amino acids 15 and 35. Regions with less pronounced hydrophobicity are found from amino acids 324 to 348 and 388 to 408.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
4.
FIG. 3

FIG. 3. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

aut5Δ cells show phenotypes characteristic of mutants defective in autophagy. (A) Total protein breakdown of aut5Δ cells during starvation is significantly reduced compared to that of wild-type cells. After metabolic pulse-labeling of growing cells with [35S]methionine, cells were shifted to 1% K acetate as a nitrogen-free starvation medium and aliquots were taken at the indicated times. The amount of acid-soluble small peptides generated via proteolysis was quantitated with a liquid scintillation counter and is expressed as a percentage of the acid-insoluble radioactivity of the 0-h sample. (B) Western blot analysis of maturation of proaminopeptidase I (pAPI) using antibodies against aminopeptidase I. mAPI, mature aminopeptidase I. Maturation is impaired in aut5-deficient cells grown to stationary phase, and this defect is complemented by expressing AUT5 from a centromeric (cen) or 2μm (2μ) plasmid.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
5.

FIG. 7. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

Immunogold electron microscopy localizes Aut5-Ha at the ER (nuclear envelope) and at ∼50-nm-diameter vesicles in the vacuolar lumen. Cells expressing Aut5-Ha from a 2μm plasmid and grown to stationary phase were starved for 4 h in 1% K acetate, fixed with paraformaldehyde, and prepared for cryosectioning as detailed in Materials and Methods. The sections were immunolabeled with antibodies to Ha and secondary antibodies coupled to 10-nm-thick gold. (A and B) pep4Δ cells. Arrows in panel A point to material enclosed in autophagic bodies. (C and D) In aut3-1 pep4Δ cells, no autophagic bodies accumulated in the vacuole due to the autophagy defect. In these cells gold particles were detected at the ER (nuclear envelope) (black arrowheads) and at ∼50-nm-diameter vesicles in the vacuolar lumen (white arrowheads). Panel B also shows localization of gold particles at ∼50-nm-diameter vesicles in the vacuolar lumen. A, autophagic body; M, mitochondrion; vm, vacuolar membrane; C, cytoplasm; V, vacuole; N, nucleus. Bar, 200 nm.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
6.
FIG. 6

FIG. 6. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

Indirect immunofluorescence microscopy suggests localization of Aut5-Ha at the ER in wild-type cells and in the vacuole and the ER in pep4Δ cells. (A to C) Cells grown to stationary phase were starved for 4 h in 1% K acetate. The cells were then fixed with formaldehyde, followed by spheroplasting with Zymolyase. The spheroplasted cells were then incubated with a primary antibody against Ha and afterwards with a secondary Cy3-coupled antibody. Nuclear DNA was further stained with DAPI. From left to right Nomarski optics (Nom), immunofluorescence microscopy (Aut5-Ha), and nuclear staining with DAPI (DNA/DAPI) are shown. (A) Wild-type cells expressing Aut5-Ha from the chromosome under the control of the endogenous promoter. (B) pep4Δ cells expressing Aut5-Ha chromosomally, which due to the lack of vacuolar endoproteinase A are defective in vacuolar protein breakdown. Arrowheads point to ER staining. (C) Chromosomal expression of Aut5-Ha in aut3Δ pep4Δ cells, which are further defective in autophagy. Bar, 20 μm. (D) Pulse-chase analysis indicates a rapid breakdown of Aut5-Ha dependent on vacuolar proteinase A. The indicated strains were grown to log phase in SMD medium, pulse-labeled for 20 min with [35S]methionine and cysteine, and chased in nonradioactive medium as described in Materials and Methods. At the specified times, aliquots were withdrawn and lysates were immunoprecipitated with antibodies to Ha. After deglycosylation with endoglycosidase H, samples were subjected to SDS-PAGE and labeled proteins were visualized using a PhosphorImager. wt, wild type.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
7.
FIG. 4

FIG. 4. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

Vacuolar acidification appears normal in aut5Δ cells. (A) The fluorescent dye quinacrine accumulates inside acidic vacuoles. In growing (upper panel) and 4-h-starved (1% K acetate) (lower panel) aut5Δ cells quinacrine accumulation indicates vacuolar acidification. vma1Δ cells, exhibiting a defect in vacuolar acidification, are included as a control. From top to bottom, Nomarski optics, fluorescence, and an overlay of both are shown. Bar, 20 μm. WT, wild type. (B) Growth of aut5Δ cells on media with pHs 5 and 7 and on medium containing 100 mM CaCl2 or glycerol as the carbon source is like that of the wild type. Cells were grown in liquid yeast extract-peptone-dextrose, and from left to right, a dilution series (1:10) of the cultures was dropped on plates and grown at 30°C. (C) Mature carboxypeptidase Y (CpY) and proteinase B (PrB) are detectable in immunoblots of growing and starved aut5Δ cells. Crude extracts of cells of the logarithmic growth phase or starved for 4 or 24 h in 1% K acetate (Ac) were subjected to SDS-electrophoresis, blotted on polyvinylidene difluoride (PVDF) membrane, and analyzed with the indicated antibodies. As a loading control, PGK was detected. p, pro; m, mature.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.
8.
FIG. 5

FIG. 5. From: Aut5/Cvt17p, a Putative Lipase Essential for Disintegration of Autophagic Bodies inside the Vacuole.

Aut5-Ha is a glycosylated integral membrane protein. (A) Aut5-Ha3 is specifically detected in immunoblots using an antibody to Ha. Crude extracts of stationary-phase cells were subjected to SDS-PAGE, blotted on PVDF membranes, and analyzed with antibodies to Ha. Lane 1, wild type; 2, wild-type (wt) cells expressing Aut5-Ha from the chromosome under the control of the native AUT5 promoter; 3, pep4Δ cells expressing Aut5-Ha from the chromosome; 4, aut5Δ cells carrying a centromeric Aut5-Ha plasmid (cen); 5, aut5Δ cells expressing Aut5-Ha from a 2μm (2μ) plasmid; 6, aut5Δ pep4Δ cells with a 2μm Aut5-Ha plasmid; 7, aut5Δ cells expressing from a 2μm plasmid a mutated version of Aut5-Ha in which serine 332 was replaced by alanine; 8, aut5Δ pep4Δ cells expressing from a 2μm plasmid a mutated version of Aut5-Ha in which serine 332 was replaced by alanine. Aut5-Ha shows a molecular mass of ∼75 kDa; note the smear of the protein to a higher molecular mass. ∗, cross-reacting material. (B) Western blotting of aminopeptidase I. The maturation of aminopeptidase I, which is similar to that of the wild type in strains expressing Aut5-Ha either from the chromosome or from plasmids, indicates the biological activity of the tagged protein (lanes 1, 2, 4, and 5). Replacement of serine 332 with alanine by site-directed mutagenesis impairs the maturation of aminopeptidase I, indicating an impaired biological activity of the mutated protein. The PVDF membrane shown in panel A was stripped and reprobed with antibodies to aminopeptidase I. pAPI, precursor aminopeptidase I; mAPI, mature aminopeptidase I. (C) Aut5-Ha is an integral membrane protein. pep4Δ cells, starved for 4 h in 1% K acetate (K-Ac), were lysed with glass beads, and the homogenate was separated by centrifugation at 100,000 × g for 45 min into pellet (P) and supernatant (S) fractions. Fractions were subjected to SDS-electrophoresis, blotted onto a PVDF membrane, and analyzed using Ha antibodies. As indicated, the cell lysate was incubated with 1 M potassium acetate, 0.1 M Na2CO3, 2.5 M urea, or 1% Triton X-100 (Tx100) prior to centrifugation. As a control, the membrane was stripped and reprobed successively with antibodies against Vph1p (an integral membrane protein), Vma2p (peripheral membrane protein), and PGK as a soluble protein. (D) Aut5-Ha is glycosylated. Cells starved for nitrogen for 4 h in 1% K acetate were lysed with glass beads; Aut5-Ha was immunoprecipitated with Ha antibodies and deglycosylated by treatment with endoglycosidase H (Endo H). After SDS-PAGE and being blotted onto a PVDF membrane, the precipitates were analyzed with Ha antibodies. Aut5-Ha∗ refers to the deglycosylated species.

Ulrike D. Epple, et al. J Bacteriol. 2001 October;183(20):5942-5955.

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