In Saccharomyces cerevisiae, surprisingly, the transmembrane protein Sbh2, which harbors an intramembrane degron, is a substrate of the ubiquitin-protein ligase Doa10.

Figure 4. Investigation of Sbh1/Sbh2 chimeric proteins. Both Sbh2 TM and ER-luminal regions contribute to the degron. (A) Schematic representation of Sbh1, Sbh2, and Sbh1/Sbh2 chimeric proteins used in this study. Sbh1-derived regions are depicted in light blue, and Sbh2-derived regions are shown in black. The nomenclature for Sbh1/Sbh2 chimeric proteins is as follows: "Sbh-XYZ" designates a chimeric protein in which the number at position X indicates the source of the cytosolic domain ("1": Sbh1; or "2": Sbh2), the number at position Y indicates the source of the TM helix, and the number at position Z indicates the source of the ER-luminal domain, respectively. (right) Comparison of Sbh1, Sbh2, and Sbh1/Sbh2 chimera TA sequences. Residues depicted bold in red represent identical residues shared between Sbh1 and Sbh2; residues depicted in light blue are Sbh1-specific residues, and residues depicted in black indicate residues specific for Sbh2; the black horizontal line indicates the TM helix, and the asterisks indicate Sbh2 residues Ser61 and Ser68, respectively. N, N terminal; C, C terminal. (B) Degradation of HA-Sbh-122. chx chase as in Fig. 1

The E6 protein of both mucosal high risk human papillomaviruses (HPVs) such as HPV-16, which have been causally associated with malignant tumors, and low risk HPVs such as HPV-11, which cause the development of benign tumors, interacts with the cellular E3 ubiquitin ligase E6AP. This indicates that both HPV types employ E6AP to organize the cellular proteome to viral needs. However, while several substrate proteins of the high risk E6-E6AP complex are known, e.g. the tumor suppressor p53, potential substrates of the low risk E6-E6AP complex remain largely elusive. Here, we report on an affinity-based enrichment approach that enables the targeted identification of potential substrate proteins of the different E6-E6AP complexes by a combination of E3-selective ubiquitination in whole cell extracts and high-resolution mass spectrometry. The basis for the selectivity of this approach is the use of a ubiquitin variant that is efficiently used by the E6-E6AP complexes for ubiquitination, but not by E6AP alone. By this approach, we identified approximately 190 potential substrate proteins for low risk HPV-11 E6 as well as high risk HPV-16 E6. Moreover, subsequent validation experiments in vitro and within cells with selected substrate proteins demonstrate the potential of our approach. In conclusion, our data represent a reliable repository for potential substrates of the HPV-16 and HPV-11 E6 proteins in complex with E6AP.

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