Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Clustal alignment of P. falciparum ( Pf ), human ( Hs ) and yeast ( Sc ) Atg8 homologs. The additional amino acids in the Apicomplexan-specific loop of Pf Atg8 are highlighted in bold. (B) Clustal alignment of P. falciparum ( Pf ), T. gondii ( Tg ) and C. parvum ( Cp ) Atg8 homologs. The Apicomplexan-specific loop is highlighted in bold red. For accession numbers of the aligned sequences see Cloning section in Materials and Methods.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Clone Assay

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Growth of Atg8-TetR/DOZI parasites expressing indicated GFP-tagged homologs from human and yeast S. cerevisiae (h and Sc, respectively) in the absence of ATc over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. Mean ± standard deviation of minimum 2 biological replicates is shown. (B) Western blots showing expression of indicated proteins during the first two cycles of the growth time course. Equal parasite numbers were loaded per lane. BiP, loading control; X, Atg8 homolog. Numbers above the blots represent parasite reinvasion cycles post ATc removal. (C) Growth of Atg8-TetR/DOZI parasites expressing Tg Atg8 or Cp Atg8 in the absence of ATc over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. Mean ± standard deviation for 2 biological replicates is shown. (D) Western blots showing Atg8 knockdown and expression of GFP- Tg Atg8 or GFP- Cp Atg8 during the first two cycles of the growth time course. Equal parasite numbers were loaded per lane. BiP, loading control. Numbers above the blots represent parasite reinvasion cycles post ATc removal.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Expressing, Standard Deviation, Western Blot

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Western blot showing membrane association of selected proteins in a Triton X-114 fractionation experiment. Numbers below the blots represent the percentage of membrane-bound GFP-tagged Atg8 homolog normalized to percentage of the membrane-bound endogenous Atg8. S, soluble fraction; M, membrane fraction; BiP, soluble protein; Atg8, membrane-bound protein; X, Atg8 homolog. (B) Representative live fluorescence images showing the localization of selected GFP-tagged Pf Atg8 homologs. DNA was stained with Hoechst 33342. Maximum intensity projections are shown. Scalebar 5 μm.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Western Blot, Fractionation, Fluorescence, Staining

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Structures of S. cerevisiae Atg8 (3VXW) and P. falciparum Atg8 (4EOY). The region corresponding to the loop in canonical homologs is in purple. The additional 9 amino acid insertion in Pf Atg8 is in green. Amino acid residues preceding the loop region are shown as blue sticks. (B) Clustal Omega alignment of S. cerevisiae (YBL078C) and P. falciparum (PF3D7_1019900) Atg8 sequences. Color code the same as in (A). Secondary structure based on Pf Atg8 structure 4EOY are indicated. (C) Schematic representation of Atg8 hybrid constructs designed for testing structural requirements for Atg8 function in malaria parasites. Light grey rectangles denote Plasmodium sequence, dark grey rectangles denote S. cerevisiae or C. parvum sequence. The sequence of the loop region is colored as in (A). Note that all constructs were N -terminally tagged with GFP for easier detection which is not shown here for clarity.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Construct, Sequencing

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: Clustal alignment of the loop region and neighboring amino acids in Atg8 homologs and putative homologs from P. falciparum ( Pf ), human ( Hs ), yeast ( Sc ), T. gondii ( Tg ), C. parvum ( Cp ), other apicomplexan parasites ( N. caninum , NCLIV_008410; S. neurona , SN3_00202075; H. hammondii , HHA_254120; C. cayetanensis , LOC34617860), and chromerids containing a secondary plastid ( V. brassicaformis , Vbra_15491; C. velia , Cvel_14496 and Cvel_25948). The additional amino acids absent from canonical homologs are in a red box.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques:

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Western blot showing membrane association of indicated Atg8 mutants in a Triton X-114 fractionation experiment. S, soluble fraction; M, membrane fraction; BiP, soluble protein; Atg8, membrane-bound protein; X, Atg8 homolog. (B) Percentage of membrane-bound Atg8 mutants normalized to the percentage of the membrane-bound endogenous Atg8. Mean ± standard deviation from 2 replicates is shown. (C) Representative live microscopy images showing the localization of indicated Atg8 mutants. DNA was stained with Hoechst 33342. Maximum intensity projections are shown. Scalebar 5 μm. (D) Quantification of parasites with the indicated localization pattern of GFP-tagged Atg8 mutants. Mean ± standard deviation of 2 independent experiments is shown.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Western Blot, Fractionation, Standard Deviation, Microscopy, Staining

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Western blot showing membrane association of Pf Atg8_RKR mutant in a Triton X-114 fractionation experiment. S, soluble fraction; M, membrane fraction; BiP, soluble protein; Atg8, membrane-bound protein; X, Atg8 homolog. (B) Representative live fluorescence images showing the localization of Pf Atg8_RKR mutant. DNA was stained with Hoechst 33342. Maximum intensity projections are shown. Scalebar 5 μm. (C) Growth of Atg8-TetR/DOZI parasites expressing GFP- Pf Atg8_RKR in the absence of ATc over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. Mean ± standard deviation for 2 biological replicates is shown. (D) Western blots showing expression of the indicated proteins in the first two cycles of the growth time course. BiP, loading control; Atg8, endogenous Atg8. Numbers above the blot represent parasite reinvasion cycles post ATc removal. (E) Growth of Atg8-TetR/DOZI parasites expressing indicated GFP-tagged Atg8 mutants in the absence of ATc over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. Mean ± standard deviation for 2 biological replicates is shown. (F) Western blots showing expression of the indicated proteins in the first two cycles of the growth time course. BiP, loading control; Atg8, endogenous Atg8; X, Atg8 mutant. Numbers above the blot represent parasite reinvasion cycles post ATc removal.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Western Blot, Mutagenesis, Fractionation, Fluorescence, Staining, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Western blot showing membrane association of indicated Pf Atg8 mutant in a Tritont X-114 fractionation experiment. S, soluble fraction; M, membrane fraction; BiP, soluble protein; Atg8, membrane-bound protein; X, Atg8 homolog. (B) Representative live fluorescence images showing the localization of the indicated Pf Atg8 mutants. DNA was stained with Hoechst 33342. Maximum intensity projections are shown. Scalebar 5 μm. (C) Growth of Atg8-TetR/DOZI parasites expressing indicated GFP-tagged Pf Atg8 mutants in the absence of ATc over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. Mean ± standard deviation for 2 biological replicates is shown. (D) Western blots showing expression of the indicated proteins in the first two cycles of the growth time course. BiP, loading control; Atg8, endogenous Atg8. Numbers above the blot represent parasite reinvasion cycles post ATc removal.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Western Blot, Mutagenesis, Fractionation, Fluorescence, Staining, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) GFP-Atg8 expression in Atg7tetR/DOZI parasites. Representative live fluorescence images of two clonal lines are shown. (B) Representative live fluorescence images of parasites expressing ATc-regulatable mCherry-Atg8 and episomal ACP L -GFP. Maximum intensity projections are shown. Scalebar 5 μm.

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

doi: 10.1101/2021.05.24.445495

Figure Lengend Snippet: (A) Growth of indicated Atg7tetR/DOZI strains expressing endogenously tagged GFP-Atg8 and the parental Atg7-tetR/DOZI strain in the absence of Atg7(-ATc), with or without IPP over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. (B) Western blot showing the expression of indicated proteins in the first two reinvasion cycles of the growth assay from (A). (C) Growth of mCherry-Atg8-tetR/DOZI in the absence of ATc, with or without IPP over 4 reinvasion cycles. Parasitemia for each time point was normalized to the culture grown in the presence of ATc. Mean ± standard deviation for 2 biological eplicates is shown. (D) Western blot showing the expression of mCherry-Atg8 in 4 reinvasion cycles of a growth assay shown in (C). Asterisk, unspecific band or degradation product of mCherry-Atg8

Article Snippet: Primary antibodies and dilutions used in this study: guinea pig anti- Pf Atg8 serum (Josman LLC), 1:1,000; mouse anti-GFP (Clontech, 632381), 1:10,000; mouse anti- Plasmodium yoelli BiP (a gift from Sebastian Mikolajczak and Stefan Kappe), 1:20,000.

Techniques: Expressing, Western Blot, Growth Assay, Standard Deviation