Figure 2. Evidence for an MBR within... | Rockefeller University Press

$Evidence for an MBR within Pbp1. (A) AlphaFold protein structure prediction of S. cerevisiae Pbp1 (AF-P53297-F1-v4) and protein domain schematic. (B) Schematic representation of Pbp1 truncation mutants assayed for their ability to interact with Mkt1 in C. The dotted lines represent the deleted regions. (C) co-IP experiments testing interactions between Pbp1 truncation variants shown in B and Mkt1. Cells expressing each mutant version of Pbp1 with Flag from the endogenous locus and Mkt1-HA were collected at log phase during growth in YPL medium. Note: Pbp1Δ299-570, Pbp1Δ491-570, and Pbp1Δ491-513 did not interact with Mkt1. (D) AlphaFold structure depicting S. cerevisiae Pbp1’s putative MBR (aa 491–549). (E) Schematic representation of Pbp1 truncation mutants assayed for their ability to interact with Mkt1 in F and G. Truncations were made within aa 491–549, which encompasses the proposed MBR. Two helical structures are predicted to reside within the MBR and are represented as orange rectangles. The Pbp1-4A strain has aa 500–503 replaced with four alanine residues. The dotted lines represent the deleted regions. (F) co-IP testing interactions between Pbp1 truncation variants depicted in E and Mkt1. Cells expressing each mutant version of Pbp1 with Flag from the endogenous locus and Mkt1-HA were collected at log phase during growth in YPL medium. Pbp1Δ500-514 and Pbp1Δ500-503 did not interact with Mkt1. (G) co-IP testing interaction between Pbp1-4A variant depicted in E and Mkt1. Cells were collected at log phase during growth in YPL medium. Pbp1-4A did not interact with Mkt1. (H) Full-length Mkt1 and His-Pbp1 (aa 482–550) were co-expressed in Rosetta cells and purified by immobilized metal ion affinity chromatography. Eluted complex was further purified by gel filtration using a HiPrep 26/60 Sephacryl S-200 High Resolution column. Depicted is the gel filtration purification trace. (I) Coomassie-stained SDS-PAGE analysis of the peak 2 fraction from H representing the complex of Mkt1 and His-Pbp1 (aa 482–550). Source data are available for this figure: SourceData F2.$

Figure 2.

Evidence for an MBR within Pbp1. (A) AlphaFold protein structure prediction of S. cerevisiae Pbp1 (AF-P53297-F1-v4) and protein domain schematic. (B) Schematic representation of Pbp1 truncation mutants assayed for their ability to interact with Mkt1 in C. The dotted lines represent the deleted regions. (C) co-IP experiments testing interactions between Pbp1 truncation variants shown in B and Mkt1. Cells expressing each mutant version of Pbp1 with Flag from the endogenous locus and Mkt1-HA were collected at log phase during growth in YPL medium. Note: Pbp1Δ299-570, Pbp1Δ491-570, and Pbp1Δ491-513 did not interact with Mkt1. (D) AlphaFold structure depicting S. cerevisiae Pbp1’s putative MBR (aa 491–549). (E) Schematic representation of Pbp1 truncation mutants assayed for their ability to interact with Mkt1 in F and G. Truncations were made within aa 491–549, which encompasses the proposed MBR. Two helical structures are predicted to reside within the MBR and are represented as orange rectangles. The Pbp1-4A strain has aa 500–503 replaced with four alanine residues. The dotted lines represent the deleted regions. (F) co-IP testing interactions between Pbp1 truncation variants depicted in E and Mkt1. Cells expressing each mutant version of Pbp1 with Flag from the endogenous locus and Mkt1-HA were collected at log phase during growth in YPL medium. Pbp1Δ500-514 and Pbp1Δ500-503 did not interact with Mkt1. (G) co-IP testing interaction between Pbp1-4A variant depicted in E and Mkt1. Cells were collected at log phase during growth in YPL medium. Pbp1-4A did not interact with Mkt1. (H) Full-length Mkt1 and His-Pbp1 (aa 482–550) were co-expressed in Rosetta cells and purified by immobilized metal ion affinity chromatography. Eluted complex was further purified by gel filtration using a HiPrep 26/60 Sephacryl S-200 High Resolution column. Depicted is the gel filtration purification trace. (I) Coomassie-stained SDS-PAGE analysis of the peak 2 fraction from H representing the complex of Mkt1 and His-Pbp1 (aa 482–550). Source data are available for this figure: SourceData F2.

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