CPY* binds to nonubiquitinated Hrd1 in nanodiscs

Using Hrd1 reconstituted in nanodiscs, binding of CPY* to non-ubiquitinated Hrd1 was investigated. Hrd1 nanodiscs were immobilized onto streptavidin magnetic beads and incubated with increasing concentrations of CPY* or CPY WT. Strikingly, CPY*

bound to non-ubiquitinated Hrd1, and the apparentKD of the interaction was approx-imately 150 nM (Figure 3.52). In contrast, CPY WT showed almost no binding to non-ubiquitinated Hrd1 across the range of Hrd1 concentrations tested. Considering that non-ubiquitinated Hrd1 in liposomes (where only the cytoplasmic side is accessi-ble) showed no affinity to CPY*, the binding to Hrd1 nanodiscs most likely originated from the luminal side of Hrd1. This result demonstrates that Hrd1 alone can differen-tiate misfolded proteins from correctly folded proteins with its luminal domain.

When Hrd1 nanodiscs were ubiquitinated on beads, the affinity for CPY* drastically increased (Figure 3.52), mirroring what was observed with ubiquitinated Hrd1 in lipo-somes (Figure 3.36). CPY WT also partially bound to ubiquitinated Hrd1 in nanodiscs, similar to what was observed with ubiquitinated Hrd1 in liposomes. The binding ob-served to ubiquitinated Hrd1 in nanodiscs is most likely from the cytosolic binding

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3.11 Binding of substrates to the luminal side of Hrd1

site, although it cannot be excluded that binding to the luminal side also occurs in the ubiquitinated state. Because data points at very low Hrd1 concentrations were missing, it was not possible to determine a reliableKD value for ubiquitinated Hrd1 in nanodiscs and CPY*.

Overall, these data indicated that CPY* most likely binds to the luminal side of Hrd1, with aK_D one order of magnitude lower than the cytosolic binding observed with ubiq-uitinated Hrd1 in liposomes (approximately 150 nM vs 7 nM, respectively. Compare Figure 3.52 with Figure 3.36). This affinity gradient for substrates from the luminal to the cytosolic side may provide the initial driving force and directionality during retrotranslocation of misfolded proteins through Hrd1 (see Discussion, section 4.6 for further details).

Figure 3.52: Differential binding of CPY* to nonubiquitinated and ubiqui-tinated Hrd1 in nanodiscs

Increasing concentrations of Hrd1 were immobilized onto streptavidin magnetic beads and incubated with 20 nM CPY* or 20 nM CPY WT (referred to as nonubiq.). Al-ternatively, Hrd1 nanodiscs were poly-ubiquitinated on beads with ubiquitination mix and then incubated with 20 nM CPY* or 20 nM CPY WT (referred to as ubiq.). The input and unbound fractions were analyzed by fluorescence scanning in a 384 well plate.

Fraction bound was quantified from the unbound fraction, normalized to the unbound fraction in the beads-only control. Shown are means ± standard deviation from three experiments. Data were fit with a single-exponential one-site binding model, from which KD for CPY* binding to nonubiquitinated Hrd1 was derived.

4 Discussion

4.1 The fusion system as a method to study retrotranslocation

This thesis concerned itself with understanding how luminal misfolded proteins get retrotranslocated by the Hrd1 complex during ERAD. Of the different classes of sub-strates degraded by the Hrd1 complex, luminal soluble misfolded proteins are of par-ticular interest because they require a protein-conducting channel spanning the entire ER membrane. As it is not clear how the Hrd1 complex accomplishes this feat, I aimed to reconstitute the process with purified components to investigate the mecha-nism of retrotranslocation in detail. Reconstitution with purified components was the method of choice, because this allows for the sufficiency of individual components to be addressed, as well as direct mechanistic details to be drawn.

A substantial amount of evidence has led to the hypothesis that the Hrd1 ubiquitin ligase forms the retrotranslocon, while the other components in the complex perform regulatory functions (Carvalho et al., 2010; Stein et al., 2014; Baldridge and Rapoport, 2016; Schoebel et al., 2017) (see section 1.9). Two previous reconstitutions of Hrd1-mediated retrotranslocation with purified components formed the basis for much of the current biochemical understanding of Hrd1. However, these studies did not faithfully recapitulate the retrotranslocation of luminal substrates due to technical reasons or experimental design (Stein et al., 2014; Baldridge and Rapoport, 2016). In the study by Stein and colleagues, a Hrd1 and CPY* complex was formed in detergent, which was coreconstituted into liposomes. Although CPY* was ubiquitinated by Hrd1 and extracted from the membrane by the Cdc48 complex, CPY* was accessible to proteases, indicating that it was not encapsulated in the lumen (Stein et al., 2014). In the study by Baldridge and Rapoport, CPY* was encapsulated into liposomes through the use of a transmembrane domain. Thereafter, the liposomes were solubilized with detergent in order to incorporate Hrd1. Strikingly, CPY* was exposed to the cytosol upon Hrd1 autoubiquitination, showing that Hrd1 retrotranslocates CPY* (Baldridge and

Chapter 4 Discussion

Rapoport, 2016). However, due to the possibility that the transmembrane domains of Hrd1 interacted with CPY* during detergent-mediated Hrd1 incorporation, it is still not clear if Hrd1 can retrotranslocate a luminal substrate “de novo” (see section 1.10.1).

To overcome these limitations, I encapsulated CPY* into liposomes and delivered it to Hrd1-containing liposomes by SNARE-mediated fusion (see section 3.1.1). This fu-sion system recapitulates retrotranslocation faithfully because it allows Hrd1 to access a substrate on its luminal side without the use of detergent. I then initiated retro-translocation by adding components of the cytosolic ubiquitination machinery, as Hrd1 autoubiquitination was demonstrated to open the channel (Baldridge and Rapoport, 2016). I monitored the ubiquitination status of the substrate as a readout for retro-translocation, since it can only be ubiquitinated when it emerges into the cytosol. I was not able to conclusively demonstrate with this system that Hrd1 can retrotranslocate luminal substrates. However, several technical limitations such as substrate aggrega-tion made it difficult to draw conclusions about the role of Hrd1 in retrotranslocaaggrega-tion.

In addition, developing the system was not trivial and required significant optimiza-tion. Several of the techniques I established, including mixing membrane proteins and encapsulated proteins via SNARE-mediated fusion, are applicable in many fields of membrane protein research. I believe that the fusion system will be a useful method for studying retrotranslocation once certain technical issues are solved. The advance-ments I made in developing the fusion system and its limitations are described in the following sections.

4.1.1 Soluble substrates can be efficiently encapsulated into