The dynamin-like protein Fzl promotes thylakoid fusion and resistance to light stress in Chlamydomonas reinhardtii
Fig 1
(a) Top. Subdomain organization of CrFzl. TP: Transit Peptide. TMP: Thiamine Mono-Phosphate synthase. HD: Potential (?) hydrophobic domain. CC: Coiled coil domain. Bottom. Phylogenetic tree of DRPs. GTPase domains of different DRPs were compared using the ClustalW online software. (b) Western Blot analysis of total cell extracts from wild-type and two distinct clones expressing transgenic 3HA-tagged CrFzl (CrFzl-HA #1 and #2) using HA and Tubulin antibodies. (c) Fluorescence microscopy of wild-type, free mVenus and CrFzl-mVenus expressing strains. Chlorophyll autofluorescence (Chl) is used as a chloroplast marker. Scale bar: 10 μm. (d) Close-up of single cells of wild-type, free mVenus and CrFzl-mVenus expressing strains. Scale bar: 5 μm. Representative images of phenotypes observed in 100% of the cells analyzed. (e) Western Blot analysis of total cell extracts from a wild-type and a CrFzl-3HA expressing strains using CrFzl and Tubulin antibodies. (f) Subcellular fractionation of chloroplasts and thylakoids from a wild-type strain. Thylakoid β-CF1 subunit of the ATPase complex (β-CF1), the large subunit of the RuBisCo (RbcL) and Nucleic Acid Binding protein (NAB1) were used as marker antibodies for thylakoid (Thy), chloroplast (Cp) and cytosol (Cyt) proteins, respectively. Loadings were adapted to reach similar amounts of β-CF1. The upper band appearing in the cytosolic fraction (asterisk) also appears in total extracts at higher exposure (See asterisk in S1b Fig). (g) Left. Western Blot analysis of the thylakoid fraction after treatment with 0, 50, 150 and 500 mM NaCl. Loadings were adapted to reach similar amounts of β-CF1. RbcL is used as a control for stromal contamination. Right. Quantification of CrFzl signal in the treated thylakoid fraction normalized with thylakoid marker β-CF1. Error bars represent the s.e.m. from four independent experiments. (h) Western Blot analysis of soluble and membrane fractions from a CrFzl-HA expressing strain. Thylakoid β-CF1 and NAB1 are used as membrane and soluble protein markers, respectively.