Figure 4. Tda2 exists both as a... | Rockefeller University Press

Figure 4.

$Figure 4. Tda2 exists both as a homodimer and in a larger complex with Aim21. (A) S. cerevisiae cytosol was fractionated by size-exclusion chromatography on a Superose 6 column, and the resulting fractions were analyzed by immunoblotting (IB) with the indicated antibodies. Recombinant Tda2 eluted at the expected position for the homodimer (peaking at fractions 19–20, Stokes radius ∼20 Å). Cytosolic cellular extracts prepared from WT cells showed endogenous Tda2 eluting as a homodimer (peaking at fractions 19–20) as well as a larger complex (peaking at fraction 13, Stokes radius ∼54 Å). Cytosolic cellular extracts prepared from tda2Δ cells portrayed no Tda2, confirming the specificity of our affinity-purified anti-Tda2 antibody. Cytosolic extracts prepared from aim21Δ cells showed the Tda2 homodimer species but no evidence of the larger Tda2 complex, indicating that Aim21 is essential for the larger Tda2 complex. In cytosolic extracts from WT cells, endogenous capping protein (Cap1) eluted in fractions 17–18. The Stokes radius of standard proteins is indicated at the top. (B) Live-cell fluorescence microscopy showing Tda2-GFP is no longer recruited to endocytic sites in aim21Δ cells. (C, left) Live-cell fluorescence microscopy showing Aim21-GFP recruitment to endocytic sites is severely reduced in tda2Δ cells. (Right) Quantification of the Aim21-GFP peak fluorescence intensity at endocytic patches in control (TDA2) and tda2Δ cells. Bars, 1 µm. Error bars indicate means ± SEM. ***, P < 0.001.$

Tda2 exists both as a homodimer and in a larger complex with Aim21. (A) S. cerevisiae cytosol was fractionated by size-exclusion chromatography on a Superose 6 column, and the resulting fractions were analyzed by immunoblotting (IB) with the indicated antibodies. Recombinant Tda2 eluted at the expected position for the homodimer (peaking at fractions 19–20, Stokes radius ∼20 Å). Cytosolic cellular extracts prepared from WT cells showed endogenous Tda2 eluting as a homodimer (peaking at fractions 19–20) as well as a larger complex (peaking at fraction 13, Stokes radius ∼54 Å). Cytosolic cellular extracts prepared from tda2Δ cells portrayed no Tda2, confirming the specificity of our affinity-purified anti-Tda2 antibody. Cytosolic extracts prepared from aim21Δ cells showed the Tda2 homodimer species but no evidence of the larger Tda2 complex, indicating that Aim21 is essential for the larger Tda2 complex. In cytosolic extracts from WT cells, endogenous capping protein (Cap1) eluted in fractions 17–18. The Stokes radius of standard proteins is indicated at the top. (B) Live-cell fluorescence microscopy showing Tda2-GFP is no longer recruited to endocytic sites in aim21Δ cells. (C, left) Live-cell fluorescence microscopy showing Aim21-GFP recruitment to endocytic sites is severely reduced in tda2Δ cells. (Right) Quantification of the Aim21-GFP peak fluorescence intensity at endocytic patches in control (TDA2) and tda2Δ cells. Bars, 1 µm. Error bars indicate means ± SEM. ***, P < 0.001.