Characteristics of Doa10-mediated ubiquitination of Sbh2

I next tested if Doa10 recognizes Sbh2 as a substrate and mediates ubiquitination in the established reconstituted system. I incubated liposomes containing Doa10, Sbh2 and Ubc6 with ubiquitination machinery (Uba1, a soluble Cue1 fragment (Cue1_sol), Ubc7, ubiquitin and ATP). To test for the requirement of Ubc6, a second set of liposomes only contained Doa10 and Sbh2. When Sbh2 is co-reconstituted with Ubc6 and Doa10, 50% of Sbh2 are polyubiquitinated (Figure 6.8). The reaction is mostly complete after 16 min (Figure 6.8B). Importantly, the decrease of the non-modified band for Sbh2 is dependent on ATP indicating that the decrease of the unmodified band is due to ubiquitination (Figure 6.8C). Interestingly, in the absence of Ubc6, no ubiquitination of Sbh2 occurs. This effect also cannot be rescued by adding a soluble fragment of Ubc6 (Ubc6_sol). This indicates that Ubc6 is required for priming Sbh2 with ubiquitin, before Ubc7/Cue1-mediated polyubiquitination can occur. In line with this, deletion of

UBC6in vivo compromises degradation of Sbh2 (Habeck et al. (2015), also see Figure 6.6). Moreover, the TM anchor of Ubc6 seems to be required for its function as an E2 enzyme.

Fraction of non-modified Ubc6 (-ATP)

+ Ubc6

Figure 6.8: Ubiquitination of Sbh2 in a reconstituted system. Liposomes containing Doa10, Ubc6DL680and Sbh2S4C-DL800or Doa10DL680and Sbh2S4C-DL800

were incubated with Uba1, Ubc7, a soluble Cue1 fragment (Cue1_sol), ubiquitin and ATP (f.c. of 0.1 µM Uba1, 1µM Ubc7, 1 µM Cue1sol, 120 µM ubiquitin, 2.5 mM ATP, 0.1µM Ubc6, 0.1µM Doa10, 0.1µM Sbh2). Where indicated, a soluble Ubc6 fragment (aa 1-231, Ubc6_sol, f.c. 1.8 µM) was present. The reaction was stopped after 0, 16, 33 and 60 min with non-reducing sample buffer. (A)Analysis of samples by SDS-PAGE and fluorescence scanning (top: DL800, bottom: DL680 fluorescence). (B, C) Quantification of (A). (B) Quantification of turnover of Sbh2. Values were normalized to 0 min timepoint. (C)Quantification of turnover of Sbh2 in reactions lacking ATP. Intensity values for 60 min timepoint were normalized to the respective 0 min timepoint.

Summarizing, we have established a reconstituted system recapitulating ubiquiti-nation of Sbh2. In the presence of Ubc6, Ubc7 and Cue1, Doa10-mediated polyubiq-uitination occurs. Our results moreover indicate that Doa10 directly recognizes Sbh2.

Thus, a minimal machinery for Doa10-mediated ubiquitination of Sbh2 is identified.

This thesis provides insight into different steps of Doa10-mediated ER-associated tein degradation (ERAD). By establishing a system to co-reconstitute membrane pro-teins by SNARE-mediated membrane fusion of liposomes, I have recapitulated ubiqui-tination of Ubc6 as well as extraction of Ubc6 from the membrane. Moreover, I have gained mechanistic insights into the extraction process by showing that Doa10 acts as a retrotranslocase. In an effort to gain further insight into structural elements in Doa10 involved in recognition and processing of Ubc6, I have tested previously described Doa10 mutants and optimized a system for site-specific crosslinking of the transmem-brane segment of Ubc6 with Doa10 (in collaboration with Iwan Parfentev, laboratory of Prof. Urlaub, MPI for Biophysical Chemistry, G¨ottingen). In order to expand our mechanistic understanding to other Doa10 substrates and to be able to draw more general conclusions, I also characterized another Doa10 substrate, the tail-anchored membrane protein Sbh2. To do so, I have first investigated the machinery for Sbh2 degradationin vivoby performing a screen (in collaboration with ´Akos Farkas, Labora-tory of Prof. Blanche Schwappach-Pignataro, University Medical Center, G¨ottingen) and also characterized this machinery by recapitulating ubiquitination of Sbh2 in a reconstituted system. In the following section, the results from these different projects are further discussed.

7.1 Co-reconstitution of membrane proteins by SNARE-me-diated membrane fusion

Reconstitution of membrane-associated processes often requires the reconstitution of multiple membrane proteins. We have established a system that allows reconstitution of membrane proteins by SNARE-mediated fusion of proteoliposomes. This system has multiple advantages compared to other reconstitution protocols. It enables high co-reconstitution efficiencies and thus makes biochemical studies possible. At the same time, it eliminates artefacts that could arise due to the interaction of membrane proteins in detergent when membrane proteins are directly reconstituted into the same set of liposomes. Moreover, it allows for a higher flexibility when two proteins are co-reconstituted whose optimal reconstitution conditions differ such as the detergent used

for solubilization of the liposomes. However, this is also a limiting factor as SNARE proteins might not be compatible with every detergent and thus reconstitution protocol.

Following, I will discuss the characteristics of the established system and compare it to other fusion approaches that have been previously developed.

7.1.1 SNARE-mediated fusion enables high co-reconstitution efficiencies High reconstitution efficiencies are an important requirement for a fusion-based co-reconstitution approach. The established SNARE-mediated fusion assay allows for high co-reconstitution efficiencies, as Ubc6 is nearly quantitatively co-reconstituted with Doa10. After fusion, pulldown of Doa10 leads to nearly complete pulldown of Ubc6 (Figure A1G). Moreover, efficient polyubiquitination and Doa10-mediated retro-translocation occur when wildtype Syb and but not when a Syb mutant (Syb∆84) is reconstituted that allows for docking of liposomes but not full fusion (Figure 3.1, 3.2 and 3.4). These two experiments indicate that the established fusion system enables high co-reconstitution efficiencies and leads to lipid-mixing of the liposome membranes.

Nordlund et al. (2014) have previously used SNARE-mediated fusion to co-recon-stitute membrane proteins of the respiratory chain with the ATP synthase (Nordlund et al., 2014). They estimate the co-reconstitution efficiency after fusion by measuring spectrophotometrically the reduction state of encapsulated cytochrome C that only upon fusion is delivered to cytochrome C oxidase. With this method, they estimate a fusion efficiency of 70%. Interestingly, they use the SNAREs SNAP-25A and syntaxin-1A which are prone to form a so called 2:1 complex consisting of two syntaxin-syntaxin-1A molecules and one SNAP-25A molecule that is inactive in fusion due to occupying the binding site for synaptobrevin (Syb) (Fasshauer et al., 1997; Margittai et al., 2001;

Xiao et al., 2001). Here, we present a strategy which uses the fusogenic ∆N-complex that consists of SNAP-25A, syntaxin-1A and a soluble Syb fragment (aa 42-96) that inhibits formation of the 2:1 complex and is displaced by full-length Syb during the fusion process (Pobbati, 2006; Hernandez et al., 2012). Our results show that using this ∆N-complex leads to high co-reconstitution of Ubc6 and Doa10 and is thus useful to co-reconstitute membrane proteins.

7.1.2 SNARE-mediated fusion allows the use of a lipid composition