Figure 6. Purified eisosome proteins... | Rockefeller University Press

Figure 6.

$Figure 6. Purified eisosome proteins from yeast structurally resemble recombinant Pil1 or Lsp1 protein assemblies. (A) Tandem affinity chromatography of tagged Pil1 enriches mainly Pil1, Lsp1, and Mrp8. Negative staining and EM reveal highly similar structures for purified eisosomes (right) as formed by recombinant Pil1 (left). Side panels show Coomassie blue–stained SDS-PAGE gels of the preparations used. (B) X-ray structure of dimeric Lsp1 BAR domain. Monomers are shown as a ribbon representation in green and gray. Residues that can be phosphorylated and that are represented in the structure of eisosome protein BAR domains are highlighted in red. (C) Purification of recombinant pil1(4D) and visualization by negative staining and EM show that pil1(4D) does not form thick helices but only long, thin filaments. Bars, 100 nm. (D) Precipitated fractions of sedimentation velocity gradients of recombinant Pil1 and pil1(4D) were analyzed by SDS-PAGE. They show different mobility of phosphorylation mutants compared with wild-type Pil1.$

Purified eisosome proteins from yeast structurally resemble recombinant Pil1 or Lsp1 protein assemblies. (A) Tandem affinity chromatography of tagged Pil1 enriches mainly Pil1, Lsp1, and Mrp8. Negative staining and EM reveal highly similar structures for purified eisosomes (right) as formed by recombinant Pil1 (left). Side panels show Coomassie blue–stained SDS-PAGE gels of the preparations used. (B) X-ray structure of dimeric Lsp1 BAR domain. Monomers are shown as a ribbon representation in green and gray. Residues that can be phosphorylated and that are represented in the structure of eisosome protein BAR domains are highlighted in red. (C) Purification of recombinant pil1(4D) and visualization by negative staining and EM show that pil1(4D) does not form thick helices but only long, thin filaments. Bars, 100 nm. (D) Precipitated fractions of sedimentation velocity gradients of recombinant Pil1 and pil1(4D) were analyzed by SDS-PAGE. They show different mobility of phosphorylation mutants compared with wild-type Pil1.