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. 2021 May 24;10:e67709. doi: 10.7554/eLife.67709

Figure 3. Simple modifications in Vps24 can be made to mimic Vps2.

(A) The domain organization of Vps2, highlighting the C-terminal region important for Vps4 binding. (B) Left panel denotes the chimeras made to replace regions of Vps2 onto Vps24. Cyan arrows in the helices are positions of the E114K mutation. Right panel represents Mup1-pHluorin sorting and canavanine sensitivity assays. In this assay, the constructs were overexpressed under a CMV promoter-Tet-off operator system.

Figure 3—source data 1. Mup1-pHluorin sorting data associated with figure in Figure 3B, Figure 3—figure supplement 1A, Figure 3—figure supplement 1B, Figure 3—figure supplement 3A, Figure 3—figure supplement 3B.

Figure 3.

Figure 3—figure supplement 1. Chimeras of Vps24-Vps2 are functional proteins.

Figure 3—figure supplement 1.

Top figure depicts the domain organization of Vps2 and Vps24. (A) Mup-pHluoring sorting assay with several chimeras of Vps24-Vps2, showing that the replacement of the C-terminal regions of Vps2 onto Vps24 keeps the constructs functional. (B) The same constructs as in (A) do not suppress vps2∆, as they are under endogeneous promoters. ‘CEN’ represents denotation for centromeric plasmid.
Figure 3—figure supplement 2. Simply adding Vps4 binding sites to Snf7 do not replace the functions of Vps24-Vps2.

Figure 3—figure supplement 2.

Canavanine sensitivity assays of Snf7 constructs with the helix-5 and MIM regions replaced from Vps2 onto Snf7. Similar replacements were also done with Snf7***, which consists of the triple mutation R52EQ90LN100I. Bottom models illustrate the idea that Vps24-Vps2 copolymer and recruitment of Vps4 through the copolymer may be required to obtain the functional heteropolymer.
Figure 3—figure supplement 3. Simple modifications in Vps24 can be made to mimic Vps2.

Figure 3—figure supplement 3.

(A) Under the endogenous promoter and centromeric plasmid (CEN), various chimeras of Vps24-Vps2 do not support the sorting of Mup1-pHluorin, but some of the same constructs when overexpressed can rescue vps24∆ (B). Note that the N-terminus of Vps2 needs to be intact to mimic Vps24. Overexpression was achieved with a CMV promoter, Tet operator system.