NAB1 | nucleic acid binding protein 1, Chlamydomonas

ODA8 distribution is similar to that of transport factors.(A) HA-tagged ODA8 expressed from an integrated transgene rescues the beat frequency (bf) of oda8 cells to wild type levels (?60 Hz), and the expressed protein migrates at ?104 kDa on blots of transformant oda8::ODA8HA (WT*) whole cell protein samples. (B) Expression of the HA-tagged transgene rescues the assembly of flagellar ODA subunit IC2 to wild type levels. Anti-IC140 recognizes inner arm I1 dynein, which is not affected by the oda8 mutation and serves as a loading control. C. Samples of whole cells (WC), deflagellated CB and flagella (FL) from equal numbers of cells (1:1) and with flagella at 50-fold excess (50:1) were probed with the indicated antibodies. ODA8HA abundance ratios are similar to those of IFT-B subunit IFT46 and IFT-associated transport factor ODA16, not those of ODA subunits such as IC2. Anti-NAB1, which recognizes a 26 kDa cytoplasmic protein, was used as a loading control in A and C.

ODA8 distribution is similar to that of transport factors.(A) HA-tagged ODA8 expressed from an integrated transgene rescues the beat frequency (bf) of oda8 cells to wild type levels (?60 Hz), and the expressed protein migrates at ?104 kDa on blots of transformant oda8::ODA8HA (WT*) whole cell protein samples. (B) Expression of the HA-tagged transgene rescues the assembly of flagellar ODA subunit IC2 to wild type levels. Anti-IC140 recognizes inner arm I1 dynein, which is not affected by the oda8 mutation and serves as a loading control. C. Samples of whole cells (WC), deflagellated CB and flagella (FL) from equal numbers of cells (1:1) and with flagella at 50-fold excess (50:1) were probed with the indicated antibodies. ODA8HA abundance ratios are similar to those of IFT-B subunit IFT46 and IFT-associated transport factor ODA16, not those of ODA subunits such as IC2. Anti-NAB1, which recognizes a 26 kDa cytoplasmic protein, was used as a loading control in A and C.

Interacting mutants alter ODA8 distribution.(A) Blots of flagella from WT* and from doublet mutant oda5, oda8 and oda10, oda8 strains that express ODA8HA (oda5* and oda10*) were probed with antibodies to HA and IC2. Both oda5 and oda10 block ODA assembly, and both greatly reduce the flagellar abundance of ODA8HA. B. Blots of flagella from WT* and from a double mutant oda6,oda8 strain expressing ODA8HA (oda6*) show that flagellar abundance of ODA8HA is not affected by the failure of ODA assembly in oda6. C. Blots of cell extracts show that cytoplasmic abundance of ODA8HA is unaffected by the oda5 and oda10 mutations. Acetylated tubulin (AcTub) is used as a loading control for flagellar samples, and NAB1 for cell extract samples. All three lanes in both blots in C. were from the same blot images. Vertical spaces indicate removal of intervening lanes.

Biochemical confirmation of AMP targeting activity. Mitochondrial, whole cell and chloroplast fractions (1 ?g protein per well) isolated from Chlamydomonas strains in which Venus localization is driven by bacillocin 1580 (A) or magainin II (B), each fused to the RBCA cleavage site, were immunolabelled with antibodies raised against FLAG, an epitope tag carried C-terminally by the Venus reporter, and markers for different cellular compartments: Cytochrome Oxidase subunit IIb (COXIIb) and ATPsynthase subunit F1? for mitochondria (mt), Photosystem II Oxygen Evolving Enhancer 2 (OEE2) and Rubisco small subunit (RBCS) for chloroplasts (cp), ?-Tubulin and nucleic-acid binding protein 1 (NAB1) for the cytosol (cyt) and luminal binding protein (BiP) for the endoplasmic reticulum (ER). Isolated chloroplasts from the Bacillocin 1580 strain (C) and isolated mitochondria from the Magainin II strain (D) were subjected to a proteinase assay, where aliquots were treated with either 150 ?g mL?1 proteinase K and/or 1% Triton X-100, a membrane solubilizing detergent. Aliquots were subsequently immuno-labelled with antibodies against FLAG, chloroplast ATP synthase subunit CF1 ? and other organelle-markers described aside.