Fig. 3
The N terminus of Rsp3 contains signals for processing and secretion. a Schematic representation of constructs used to analyze N-terminal processing of Rsp3. In all constructs, expression was driven from the constitutive otef promoter and all contain a C-terminal HA-tag. Amino acid sequences from 24 to 70 downstream of the signal peptide (SP) are shown. The position of Myc-tag insertion is depicted in light blue. Amino acids DGGA identified by N-terminal sequencing as N terminus of secreted Rsp3 are indicated in red. The arrow indicates the cleavage site. Amino acids subjected to alanine substitution are indicated in green. b Western blot analysis of Rsp3 secretion. SG200Δrsp3 strains expressing the indicated proteins were grown in CM liquid medium to an OD600 of 0.6. Proteins from cell pellets and from supernatants (collected after TCA precipitation) were prepared subjected to western blot. The western blots were developed with either anti-HA or anti c-Myc antibodies as indicated. Detection of tubulin via an anti-tubulin antibody served as internal control for a cytosolic protein. c Secretion of Rsp3 variants carrying amino acid substitutions or deletions in the N-terminal domain. SG200Δrsp3 strains expressing the indicated proteins were analyzed by western blot after fractionation in supernatant and cell pellet as in b. Proteins shown in western blot analysis are derived from two independent clones. Experiments were repeated two times for Fig. 3b and three times for Fig. 3c, and one representative experiment is shown. Full blots are shown in Supplementary Fig. 11
