A. Western blot analysis of aminopeptidase I (Ape1) in yeast mutant atg8Δ (lane 1), wild type (lane 2) and yeast mutant atg12Δ (lane 3) S. cerevisiae. Cells were incubated under starvation conditions in SD(-N) medium for 16 h, lysed, and API processing was analyzed using rabbit α-Ape1 antibodies. ProApe1 and mature Ape1 (mApe1) are indicated.
B. Western blot analysis of Ape1 in atg8Δ yeast transformed with pCM185 containing L. major ATG8 homologues: ATG8 (lane 3), ATG8A (lane 4), ATG8B (lane 5), ATG8C (lane 2) or transformed with pCM185 only (lane 6). Wild type S. cerevisiae are shown in lane 1. The cells were cultured under starvation conditions in SD(-N) medium for 4 h and Ape1 processing was analyzed using rabbit α-API antibodies. ProApe1 and mature Ape1 (mApe1) are indicated.
C1. Amino acid sequence alignment of the C-terminal end of L. major ATG12 with those of T. brucei, S. cerevisiae, human and A. thaliana. Identical and conserved amino acids are shaded in black and grey, respectively. Shade of grey depends upon number of residues conserved. The carboxyl-terminal glycine identified in A. thaliana ATG12 (Hanada et al., 2005) and the two hydrophobic residues (Y¹⁴⁹ and F¹⁵⁴) required for conjugation to ATG10 (Suzuki et al., 2006; Hanada et al., 2006) and essential for the formation of the ATG12-ATG5 conjugate are marked with an asterisk. L. major, LmjF22.1300; T. brucei, Tb927.7.3320; S. cerevisiae, P38316; human, AAH11033; A. thaliana, AAM70187. The numbers in parenthesis represent the amino acid number of the first residue in the alignment.
C2. Western blot analysis of Ape1 in atg12Δ S. cerevisiae transformed with pCM185 containing L. major ATG8 homologues: ATG8 (lane 3), ATG8A (lane 4), ATG8B (lane 5), ATG8C (lane 6), the putative L. major ATG12 (lane 7) or transformed with pCM185 only (lane 2). Wild type S. cerevisiae were used as the control (lane 1). The S. cerevisiae were cultured under starvation condition for 16 h and Ape1 processing was analyzed using a rabbit α-API antibodies. ProApe1 and mature Ape1 (mApe1) are indicated.
D. Western blot analysis of Ape1 in atg5Δ S. cerevisiae transformed with pCM185 containing L. major ATG5 (lane 3) and pCM185 only (lane 2) and treated as described in C2. Wild type S. cerevisiae was included as a positive control (lane 1).
E. Western blot analysis of Ape1 in atg10Δ S. cerevisiae transformed with pCM185 containing the L. major ATG10 homologue (lanes 2-3), or transformed with pCM185 only (lane 1) or ATG8 (lane 5) and treated as described in C2. Wild type S. cerevisiae was included as positive control (lane 4).
F. Western blot analysis of ApeI in atg8Δ S. cerevisiae (lanes 2-4) and atg12Δ S. cerevisiae (lanes 5-7) transformed with pCM185 containing ATG8_g (lanes 2, 5), ATG12_g (lanes 3, 6) and S. cerevisiae ATG12 (lanes 4, 7), respectively, and treated as described in C2. Wild type S. cerevisiae was included as positive control (lane 1).

A. The occurrence of punctate structures in live L. major promastigotes containing GFP-ATG8 (row 1), GFP-ATG8A (row 2), GFP-ATG8B (row 3) and GFP-ATG8C (row 4). Promastigotes expressing GFP-ATG8A were visualised after 4 h incubation in nutrient-deprived medium. Arrowheads indicate punctate structures. Scale bar, 10 μm.
B. Quantification of punctate structure formation in promastigotes after incubation for 4 h in nutrient-deprived medium. The number of promastigotes containing at least one GFP-ATG8 punctum is expressed as a percentage of all GFP-expressing cells. The data are means ± SE from three series of measurements from three independent experiments. *, data for GFP-ATG8 and GFP-ATG8A cell lines after starvation differed significantly from those for cells maintained in nutrient-rich medium (p<0.01).
C. The appearance of live Δatg4.2 promastigotes containing GFP-ATG8A (C1) or GFP-ATG8 (C2) after 4 h starvation in nutrient-deprived medium. Arrowheads indicate puncta. Scale bar, 10 μm.

A. Soluble fractions of E. coli expressing His-ATG8-HA (row 1), His-ATG8A-HA (row 2), His-ATG8B-HA (row 3), His-ATG8C-HA (row 4) and His-ATG12-HA (row 5) alone (lane 1) or co-expressed with either ATG4.1 (lane 2) or ATG4.2 (lane 3) were incubated for 30 min at 30°C in 50 mM Tris-HCl, pH 8.0, 125 mM NaCl and 40 mM β-mercaptoethanol and then analysed by Western blot with α-His antibody. The cleaved ATG8 bands are labelled with an asterisk.
B. Hydrolysis of His-ATG8-HA by ATG4.1.
(B1) The hydrolysis was inhibited by 1 mM NEM (lane 2) and 1 mM iodoacetamide (lane 6) but unaffected by 1 mM PMSF (lane 3), 1 mM pepstatin (lane 4) and 10 mM 1,10-phenanthroline (lane 5). Lane 1 shows the control (no inhibitor added). Incubation was as described in A.
(B2) Hydrolysis of His-ATG8-HA by ATG4.1 was progressive over 60 min.
C. Analysis of the product of the cleavage of His-ATG8-HA by ATG4.1. Hydrolysis was over 30 min as described in A.
(C2) The product was purified by Ni⁺-affinity chromatography and analysed by SDS-PAGE, two main proteins were apparent.
(C1) The cleaved products were analysed by MALDI TOF. Six molecules were detected and their molecular masses are given.

A. Occurrence of RFP-ATG12-containing structures (A2) and GFP-ATG8-containing putative autophagosomes (A1) in wild type L. major promastigotes expressing both proteins. The position of the kinetoplast is shown (blue) using DAPI staining. L. major procyclic promastigotes were suspended in nutrient-deprived medium for 120 min and observed by fluorescence microscopy. The merge of the RFP-ATG12 and GFP-ATG8 images, the co-labelled structure is enlarged at the bottom left, and the DIC image of the promastigote are shown in A3 and A4, respectively. Scale bar, 10 μm.
B. Western blot analysis using α-GFP (B1) and α-RFP (B2) antibodies on L. major procyclic promastigote cell extracts (5-8 × 10⁶ cells ml⁻¹) transfected with GFP-ATG8 and RFP-ATG12. The α-GFP antibody detected a faster migrating lipidated ATG8 (labelled GFP-ATG8-PE) and a higher molecular mass unlipidated ATG8 (labelled GFP-ATG8*), resolved in a urea SDS-PAGE gel (B1). The anti-RFP antibody showed just a single protein (labelled RFP-ATG12) consistent with the predicted size of the fusion protein (B2).

A. Western blot analysis using α-GFP antibody on Δatg4.2 [pN-ATG8] promastigote cell extracts (5-8 × 10⁶ cells ml⁻¹) show an abundance of lipidated ATG8 (labelled GFP-ATG8-PE) (lane 0). Incubation of the cell extract with recombinant ATG4.2 for 90 and 180 min at 30°C resulted in a decrease in GFP-ATG8-PE (lanes 2 and 3). Transketolase was used as an internal loading control.
B1. WT[pN-ATG8] (open squares) and WT[pN-ATG4.2/ pN-GFP-ATG8] (closed circles) promastigotes were compared for the occurrence of putative autophagosomes during growth in vitro in normal medium. Data are means ± SD from 3 independent experiments.
B2. Western blot analysis of extracts from early stationary phase WT[pN-ATG4.2/pN-GFP-ATG8] (lane 1) and WT[pN-GFP-ATG8] (lane 2) promastigotes separated by SDS-PAGE containing 6 M urea and detected with α-GFP antibody. Cleaved ATG8 (GFP-ATG8*) and lipidated ATG8 (GFP-ATG8-PE) are indicated. EF1α was used as an internal loading control.
C. Sensitivity to starvation of WT[pN-ATG8] (open squares) and WT[pN-ATG4.2/pN-GFPATG8] (closed circles) L. major promastigotes. Cells were incubated in PBS and their viability assessed by the MTT assay. Data are means ± SD from four replicates. *: data for WT[pN-ATG4.2/pN-GFP-ATG8] and WT[pN-GFP-ATG8] promastigotes differed significantly (P < 0.05).