FIGURE 5.
Protein O-mannosylation at the post-translational translocon. A, signal peptides of prepro-α-factor (ppαF), native Ccw5, and coCcw5. The hydrophobic core sequences are shown in italics and sites of signal peptidase cleavage are indicated by dashes. The Suc2 sequence of the chimeric Ccw5 fusion protein (coCcw5) is underlined. B, analysis of the hydrophobicity of the signal peptides of ppαF, Ccw5, and Suc2. The average hydrophobicity of the hydrophobic core sequences was calculated as described previously (35). The window is defined as the amino acids that follow the last positively charged residue of the n-region that precedes the hydrophobic core. C, left, scheme of experimental workflow. Right, in vitro translation of mRNAs coding for Ccw5FLAG constructs (lanes 1, 3, and 4) in the presence of WT yM (lanes 2 and 5–7). Post-translational translocation is indicated by the presence of CHX. The translation product of ppαF mRNA is indicated. After anti-FLAG precipitation, proteins were separated by SDS-PAGE, and analyzed by autoradiography.