Figure 9. Rop^G11 abolishes the biochemical interaction between Rop and syx1A and rescues homeostasis defect in Rop.

(A) Schematic of the Drosophila Rop protein. Point mutation Rop^G11 is indicated by red star at syntaxin-binding domain (SBD) of the Rop protein shown in pink. Rop^G11 converts Aspartic Acid (D45) to Asparagine (N). This site is conserved in mammalian Rop (munc18-1). (B) Coomassie stains of in vitro binding assays. (left) MBP-Rop (110 kDa) coprecipitated with bead-bound GST fusions of syx1A (GST-syx1A^ΔC) (60 kDa). MBP-Rop does not bind to GST-syx1A^ΔC in the presence of single point mutation at the N-terminal of Rop (MBP-Rop^G11). (right) Free MBP-Rop in the absence of GST-syx1A. (C) Binding curves quantify dissociation constant (K_d) for MBP-Rop and MBP-Rop^G11 binding to GST-syx1A^ΔC; x-axis is concentration of MBP recombinant protein used (μM); y-axis is the fraction of protein bound; n = 2 (D) Sample traces showing EPSC amplitudes ± PhTx for Rop^G11 heterozygous null allele (Rop^G11/+), heterozygous Rop^G11 placed in trans with Rop^G27 mutant (Rop^G27/RopG^G11), and heterozygous Rop^G11 placed in trans with Df^Rop (Df^Rop/RopG^G11). (E) Average data for mEPSP amplitude ±PhTx for indicated genotypes. PhTx application reduces amplitude in all genotypes (p<0.01). Data represent mean ±SEM. Student’s t test. two tailed. (F) Average data for EPSC amplitude as in (B); ns, not significant; Student’s t test, two tailed.