Next, I tested if carboxy-terminal fluorescent labels attached to Ubc6 affect its be-haviour. To do so, Ubc6 was carboxy-terminally labeled by sortase-mediated transpep-tidation (Popp et al., 2009). Different Ubc6 variants were thereby generated that were labeled with the fluorescent dyes Dylight680 (DL680), DyLight800 (DL800) or Alex-aFluor488 (A488). The behaviour of these Ubc6 variants was compared to a Ubc6 construct that does not contain any carboxy-terminal label. It instead is labeled at the cytosolic side at its active-site cysteine with A488 via maleimide chemistry (Ubc6C87-A488). I then tested if Doa10 releases these Ubc6 variants.
I co-reconstituted these Ubc6 variants with t-SNARE and fused them to Doa10, Syb liposomes. I then tested for Doa10-mediated release of Ubc6 by incubation with the chaperone Get3, as in Figure 3.4A and 3.4B. After incubation with Get3, liposomes were immobilized via co-reconstituted biotinylated lipids and samples of input and supernatant analyzed by SDS-PAGE. About 45% of Ubc6C87-A488 were detected in the supernatant (Figure 4.5C). A similar fraction of Ubc6DL680and slightly more Ubc6A488 were released (Figure 4.5B and 4.5D). In contrast, less Ubc6DL800 (about 30%) was detected in the supernatant (Figure 4.5A). Concluding, a C-terminal fluorescent label on Ubc6 can interfere with the release assay, either by interfering with Doa10-mediated release or with binding of Get3 to the TM anchor of Ubc6. Different fluorescent labels have thereby different effects. Whereas C-terminal labeling of Ubc6 with DL680 does not influence the assay, as its behaviour is comparable to Ubc6 labeled at the cytosolic side, labeling of Ubc6 with A488 increases and labeling with DL800 decreases the amount of Ubc6 detected in the supernatant.
Interestingly, the release efficiency correlates with the molecular weight of the C-terminal label. A488 is the smallest label I tested (721 g/mol), whereas DyLight800 is larger (1075 g/mol) (see Table 4.1). This, probably combined with differences in hydrophobicity, may lead to the observed differences in release efficiency. For experi-ments, I have used Ubc6 labeled with A488 or DL680, unless otherwise indicated.
Table 4.1: Molecular weight of A488, DL680 and DL800 maleimide.
Values for molecular weight obtained from Thermo Fisher Scientific.
Dye Molecular weight (g/mol)
AlexaFluor488 C5 maleimide 721
DyLight680 maleimide 972
DyLight800 maleimide 1075
0.0 0.2 0.4 0.6
-Get3 +Get3 -Get3 +Get3 -Get3 +Get3 +Doa10 Inhibited -Doa10
Ubc6DL680 Ubc6DL800 Ubc6C87-A488 Ubc6A488
Released fraction of Ubc6 (Supernatant / Input)
Get3 - + - + - ++Doa10Inhibited-Doa10
- + - + - ++Doa10Inhibited-Doa10 Input Supernatant
Get3 - + - + - ++Doa10Inhibited-Doa10
- + - + - ++Doa10Inhibited-Doa10
Get3 - + - + - ++Doa10Inhibi ted
-Doa10
- + - + - ++Doa10Inhibited-Doa10
Get3 - + - + - ++Doa10Inhibited-Doa10
- + - + - ++Doa10Inhibited-Doa10 Ubc6800
Ubc6C87-A488 Ubc6A488
35 35
Input Supernatant
Input Supernatant Input Supernatant
Ubc6DL680
A B
C D
E
0.1 0.3 0.5
Figure 4.5: Influence of the C-terminal fluorescent label of Ubc6 on sub-strate behaviour. Different Ubc6 variants (Ubc6DL680, Ubc6DL800, Ubc6A488, Ubc6C87-A488) were co-reconstituted with t-SNARE and the liposomes fused with liposomes containing Doa10 and Syb. Liposomes were subsequently incubated with Get3 (f.c. 10µM Get3, 40 nM Doa10, 100 nM Ubc6). Liposomes were im-mobilized via co-reconstituted biotinylated lipids. Samples of the supernatant and input were analyzed by SDS-PAGE and fluorescence scanning, for(A)Ubc6DL680, (B) Ubc6DL800, (C) Ubc6C87-A488, (D) Ubc6A488. (E) Quantification of (A-D).
Fraction of Ubc6 in supernatant relative to input was quantified.
for Doa10 function
Doa10 has a TM region comprising 14 TM segments which contains the conserved TD-domain (TM segments 5-7). Studies indicate that this TM region is functionally important. First, mutation of conserved residues in TM segment 5 affects degradation of ERAD substrates (Kreft and Hochstrasser, 2011). Moreover, it has been shown that Doa10 recognizes an intramembrane degron in the substrate Sbh2 (Habeck et al., 2015). The Doa10 TM region might be important for recognition of such a membrane-localized degradation signal. Moreover, it has been hypothesized that the TM domain of Doa10 might have a role in substrate retrotranslocation.
Our established reconstituted system provides some insight into the role of the TM region of Doa10. It recapitulates recognition, ubiquitination and retrotranslocation of Ubc6. In this system, a Doa10 truncation containing only the RING domain and the TM segments 1 and 2 (Doa101-468) is less efficient in Ubc6 ubiquitination compared to full-length Doa10 and is not able to act as a retrotranslocase for Ubc6 (see Figure 3.5).
These results indicate that the TM region of Doa10 is important for ubiquitination as well as retrotranslocation of Ubc6. The TD-domain might thereby play a role.
Kreft and Hochstrasser identified Doa10 variants that contain a mutation in the TD-domain and affect degradation of Ubc6 (Kreft and Hochstrasser, 2011). I therefore wanted to test how these Doa10 mutants behave in our reconstituted system. I also sought to identify interaction sites between Ubc6 and Doa10 and therefore optimized a protocol for site-specific photocrosslinking of Ubc6 with Doa10.
5.1 Characterization of Doa10 variants containing mutations in the TD domain
Different Doa10 TD mutants have been described that impair degradation kinetics of Ubc6. Doa10 contains a conserved glutamate residue at position 633. Interestingly, mutating this glutamate to glutamine (E633Q) results in enhanced degradation of Ubc6. In contrast, the degradation of Ubc6 is slowed down when the charge is preserved (E633D). Moreover, these mutations of E633 specifically affect degradation of Ubc6, but not of the soluble substrate Deg1-Ura3 or the membrane-bound
Deg1-Vma12-Ura3 (Kreft and Hochstrasser, 2011).
Impaired degradation kinetics of Ubc6 upon mutation of Doa10 can be the result of a change in Doa10-mediated ubiquitination of Ubc6 in multiple ways. First, interaction of Ubc6 with the ubiquitin ligase can be affected and thus affect substrate recognition as well as ubiquitination. Moreover, during ubiquitination of Ubc6, Doa10 does not only have to bind to substrate, but also coordinate the E2 enzyme Ubc7 with its cofactor Cue1. Altered coordination of the E2 enzyme by the ubiquitin ligase might affect substrate ubiquitination and thus degradation kinetics. In addition, mutation of Doa10 might affect its interaction with other substrates and thus indirectly affect degradation of Ubc6. I directly tested the behaviour of these Doa10 mutants in our established reconstituted system. I first analyzed their behaviour in ubiquitination of Ubc6.
Interestingly, Ubc6 is ubiquitinated by these Doa10 mutants with similar kinetics compared to wildtype Doa10 (Figure 5.1A, B). The ubiquitin chain lengths created only show minor differences. In the presence of Doa10E633D or Doa10E633Q slightly more monoubiquitinated and concomitantly less polyubiquitinated Ubc6 is generated, compared to Doa10 WT (Figure 5.1C). These small differences might not necessarily be due to the mutation itself, but due to other factors such as small differences in protein concentration, purity or in general variability between different preparations of Doa10. Concluding, I do not observe major differences in Ubc6 ubiquitination in the presence of the Doa10E633 mutants.
180 Doa10 WT Doa10E633DDoa10E633Q no Doa10
Ubc6DL800
Figure 5.1: Ubiquitination of Ubc6 in the presence of Doa10E633D and Doa10E633Q. Liposomes containing Ubc6DL800 and t-SNARE were fused with liposomes containing Doa10, Cue1 and Syb (lipid:protein ratios of 5000, 2000 and 2000, respectively) and subsequently incubated with ubiquitination machinery (f.c. 0.1µM Uba1, 0.5 µM Ubc7, 60 µM ubiquitin, 0.2 µM Ubc6, 80 nM Doa10, 0.2µM Cue1). (A)Analysis of samples by SDS-PAGE and fluorescence scanning.
(B) Quantification of Ubc6 turnover. (C) Analysis of ubiquitin chain length (60 min timepoint).
Different degradation kinetics in vivo could not only be a result of affected ubiq-uitination, but also different extraction efficiencies. As I observed that Doa10 is a retrotranslocase for Ubc6 (Chapter 3), I wanted to therefore test next if the de-scribed Doa10 mutants are capable of retrotranslocating Ubc6 in our reconstituted system. Besides Doa10E633D and Doa10E633Q, I also tested two other Doa10 mu-tants which affect degradation of Ubc6, but also the Doa10 substrate Deg1-Ura3 (Doa10P638A G642A and Doa10G636R, (Kreft and Hochstrasser, 2011)). Liposomes
con-taining these Doa10 mutants or Doa10 WT and Syb were fused with liposomes contain-ing Ubc6A488 and t-SNARE. Doa10-mediated release was again measured by monitor-ing the A488-fluorescence upon anti-A488 addition. All tested Doa10 mutants released Ubc6 with similar kinetics compared to wildtype Doa10 (Figure 5.2).
0.0 0.5 1.0
0 10 20 30 40 50 60 70 80
Doa10 WT - Doa10
Doa10E633D Doa10E633Q Doa10P638A G642A
Doa10G636R
Time (min)
A488-fluorescence (a.u.)
+ab
+det
Figure 5.2: Release of Ubc6 by Doa10 TD mutants. Liposomes contain-ing Ubc6A488 and t-SNARE were fused with liposomes containing Doa10 (WT, E633D, E633Q, P638A G642A, or G636R) and Syb, and the A488 fluorescence was subsequently measured upon anti-A488 addition (+ab). Where indicated, detergent (+det) was added to solubilize liposomes.
Concluding, I do not observe major differences in ubiquitination or retrotransloca-tion of Ubc6 in the presence of described Doa10 mutants harboring a mutaretrotransloca-tion in the TD-domain.