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. 2018 Apr 16;293(22):8362–8378. doi: 10.1074/jbc.RA118.002457

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© 2018 Martins et al.

Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

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Figure 6. — Biochemical and in vivo determination of the Ssy5 autolytic cleavage site. A, Ssy5 from S. cerevisiae and orthologs from the related fungi were compared using sequence alignment algorithm AlignX (Vector NTI). The level of similarity of the aligned proteins is plotted; numbers correspond to amino acid residues of the longest ortholog (Lodderromyces elongisporus). The sequence alignment of the region containing the putative autoprocessing site (17) (scissors; between residues 381 and 382 in S. cerevisiae) is shown expanded below the plot. Identical (yellow), conservative (blue), and similar (green) residues are highlighted; residues with weak (green text) or no similarity (black) are indicated. B, mass spectrometric analysis of the Ssy5 pro- and Cat domain. Ssy5 was purified from lysates from CAY324 (ssy5Δ) carrying pCA260 (HAi-SSY5) grown in YPD. HAi-Ssy5 has a 3xHA tag (HAi) inserted internally in the prodomain and has GST fused to the C terminus of the Cat domain. Coomassie staining revealed two prominent species corresponding in size to the Cat domain (band I) and the prodomain (band II). The bands were subjected to trypsin digestion, and resulting peptides were analyzed by MS. The Ssy5 protein sequence with peptides recovered from band I and band II are highlighted in red and orange, respectively. The diagnostic peptides identified, i.e. the most C-terminal peptide of the prodomain and the most N-terminal peptide of the Cat domain are underlined (solid line). Amino acids DYIKKA (dashed underline) were not identified. C, in vivo analysis of Ssy5 autolysis. Schematic presentation of Ssy5 constructs; the positions of the 3xHA tag (HAi), autolytic processing site (between Ala-381 and Ala-382), FLAG-tag (inserted after Ala-382), and C-terminal GST fusion are indicated. Growth of strain PLY1351 (ssy5Δ) transformed with pRS316 (vc), pCA260 (HAi-Ssy5), or pTP103 (HAi -Ssy5-FLAG) were spotted on SD and SD medium containing AzC (right panel). Immunoblot analysis of extracts from the strains (lane 1, vc; lane 2, HAi-Ssy5; lane 3, HAi-Ssy5-FLAG) grown in SD (lower panel). The immunoreactive forms of Ssy5 are represented at their position of migration; the parentheses around the FLAG-tag symbol indicates that it is uniquely present in extracts in lanes 3. The position of unrelated cross-reacting proteins is marked with stars. Molecular markers (kDa) are indicated at the position of migration (left of immunoblots).