Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy

Thank you for submitting your work entitled "Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy" for peer review at eLife. Your submission has been favorably evaluated by Randy Schekman (Senior editor and Reviewing editor) and three reviewers.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

This work describes a biochemical approach to investigate the relationship between p62 oligomerization and cargo binding. Contrary to Atg19, a substrate adaptor with multiple LIR motifs, p62 contains only a single LIR-motif and a single UBA-domain. This study suggests that oligomerization provides p62 with multiple LIR-motifs and UBA domains within a single structure, probably related to the large helical structures previously described by cryo-EM. Oligomerization decreases the off-rate of cargo from the adaptor, thus stabilizing the interaction of adaptor and cargo sufficiently for membrane formation around the cargo. In addition, the simultaneous binding of p62 to ubiquitin and LC3 allows for bending of the autophagosomal membrane around cargo, at least in the context of GUVs. There is also some correlation between in vitro data and recognition of ubiquitylated Salmonella by p62.

Overall, there are several interesting observations in this manuscript, that paired with careful biochemical characterization make it an interesting study for publication in eLife.

Essential revisions:

1) My major concern with this study is the ubiquitin aspect: cargo for p62 is usually heavily ubiquitylated, with either long chains or more complex ubiquitin structures being observed most frequently. However, this study is mostly done in the context of mono- or di-ubiquitin, and linkage-specificity of ubiquitin conjugates is not taken into account. It is important to address both length and linkage of the ubiquitin conjugates as previous work had suggested that long ubiquitin chains (octamers connected through K63) can disrupt the oligomeric structure of p62. Thus, some of these assays need to be repeated with defined K63- or K48-linked chains of at least four ubiquitin molecules tested for their interaction with p62 and their ability to induce membrane bending in conjunction with p62.

2) The assay in Figure 5D-F is nice. Are all components required for bending the membrane or that an interaction between the beads and the membrane could be sufficient to drive the curvature? The authors may need to demonstrate a physiological circumstance which requires ubiquitin, oligomerized p62 and LC3 to bend the membrane. Could the p62-coated beads bend the LC3-coated membrane? In Figure 5F, the data shown do not support the statistical significance of the claim that "The oligomerization deficient mutants of p62 showed reduced ability to mediate membrane bending (Figure 5F)." The authors show that PB1-mediated oligomerization is important for avid binding to Ub, but their results do not support the idea it is a direct driving force for membrane bending.

3) Figure 6F-G do not appear to show significant effects. In 6G in particular, the effects are very small. This is probably due to adaptor redundancy. The authors speculate about other adaptors that might be involved. On balance the data in Figure 6F-G neither support nor oppose the authors' hypothesis.

4) Figure 7C. While the histogram shows what looks like an impressive reduction for the PB1 mutants, I'm confused about how colocalization was scored. By eye it looks like plenty of GFP is colocalized with all of the mutants in Figure 7A.

5) The advance of the study is to illustrate the enhanced association of p62 with the LC3 and ubiquitin by oligomerization. However, the process is insufficiently characterized. The reviewer suggest calculating a binding curve between the lipid anchored LC3 and different concentrations of different p62 variants. Further, the binding kinetics in addition to the dissociation kinetics indicated in Figure 3 may also be helpful. If oligomerization is just simply increasing the affinity to LC3 and ubiquitin binding, could an engineered p62 with multiple LIFs fulfill similar purpose?

6) Oligomerization decreases the diffusion rate of p62 in the cytosol which may be a negative contributor to its dynamics and efficiency to recognize cargo. Therefore, it is possible that the extent of p62 oligomerization is low but enhanced by cargo binding or association with LC3. Could the author examine the effect of ubiquitin and LC3 on the extent of p62 oligomerization?

7) The author may consider revising the conclusion in the subsection "Oligomerization of p62 renders binding to LC3B-coated surfaces irreversible". The authors indicated the "concentrated p62". Here it is difficult to conclude from Figure 3F-H that these soluble LC3 is not concentrated, as once they associate with the oligomerized p62 concentrated on the beads, they may become concentrated too. The reviewer suggests modifying "concentrated" to "membrane associated". Otherwise, it is necessary to quantify the fluorescence intensity of the LC3 on the lipid surface as well as those associated the p62-coated beads to make a comparison. In addition, the authors may consider titrating the amount of LC3 on the membrane and quantifying the dissociation rate of oligomerized p62 from the membranes coated with different concentrations of LC3 proteins.

1) My major concern with this study is the ubiquitin aspect: cargo for p62 is usually heavily ubiquitylated, with either long chains or more complex ubiquitin structures being observed most frequently. However, this study is mostly done in the context of mono- or di-ubiquitin, and linkage-specificity of ubiquitin conjugates is not taken into account. It is important to address both length and linkage of the ubiquitin conjugates as previous work had suggested that long ubiquitin chains (octamers connected through K63) can disrupt the oligomeric structure of p62. Thus, some of these assays need to be repeated with defined K63- or K48-linked chains of at least four ubiquitin molecules tested for their interaction with p62 and their ability to induce membrane bending in conjunction with p62.

Thank you very much for this important point. We have now tested the interaction of wild type p62 and the non-oligomeric delta PB1 mutant with beads cross-linked to linear tetra-ubiquitin, K48-linked tetra-ubiquitin and K63-linked tetra-ubiquitin (Figure 4E). Interestingly, while the delta-PB1 mutant bound equally to all three chain types, the oligomeric wild type protein showed increased binding to linear ubiquitin and to some extent to K63-linked ubiquitin chains but not to K48-linked ubiquitin. When we tested the oligomer disruption activity of the three chain types we found that K48-linked ubiquitin chains had the strongest effect on oligomerization (Figure 4F). Thus it appears that K48-linked ubiquitin chains may not be the preferred target for oligomeric p62. We further tested the effect of ubiquitin chain length on p62 binding using mono-ubiquitin and linear di- and tetra-ubiquitin (Figure 4G). Both, wild type and p62 delta PB1 bound stronger to longer chain types but the positive effect of the increasing chain length was more pronounced for the oligomeric wild type protein. This was despite the fact that at high concentrations linear ubiquitin chains have a considerable disruptive effect on p62 oligomerization (Figure 4H). We then went on to test the dissociation of p62 from mono-ubiquitin and linear tetra-ubiquitin using FRAP (Figure 4I) and found that p62 is much more tightly bound (i.e. has a lower off rate) to tetra-ubiquitin chains than to mono-ubiquitin. Increasing the density of mono-ubiquitin on the beads also resulted in reduced off-rates of p62 from the beads (Figure 4J). Thus a major factor for the avid binding of oligomeric p62 to ubiquitinated structures is the density of ubiquitin rather than the existence of chains, although chains could still have an additive positive effect. We also tested the ability of wild type p62 to bend the membrane of GUVs around beads coated with linear tetra-ubiquitin, K48-linked tetra-ubiquitin and K63-linked tetra-ubiquitin (Figure 5F). We found that p62 mediated membrane bending for all three chain types. Consistent with weaker interaction of p62 with K48-linked chains bending was less efficient for this chain type.

2) The assay inFigure 5D-Fis nice. Are all components required for bending the membrane or that an interaction between the beads and the membrane could be sufficient to drive the curvature? The authors may need to demonstrate a physiological circumstance which requires ubiquitin, oligomerized p62 and LC3 to bend the membrane. Could the p62-coated beads bend the LC3-coated membrane? In Figure 5F, the data shown do not support the statistical significance of the claim that "The oligomerization deficient mutants of p62 showed reduced ability to mediate membrane bending (Figure 5F)." The authors show that PB1-mediated oligomerization is important for avid binding to Ub, but their results do not support the idea it is a direct driving force for membrane bending.

We have now tested beads directly cross-linked to p62. In order to prevent massive crosslinking of beads by oligomeric p62 we have used delta PB1 and the corresponding LIR mutant for this experiment. We found that cross-linked p62 delta PB1 efficiently mediated membrane bending (Figure 5E) showing that the presence of ubiquitin per se is not essential for the membrane bending activity of p62. In addition, this experiment shows that oligomerization is also not essential for membrane bending (although it could of course have an additive positive effect). Thus oligomerization may primarily mediate the accumulation of p62 at the ubiquitinated cargo, which in turn clusters the LIR motifs allowing the bending of the LC3B-coated membrane. We thank the reviewers for suggesting this insightful experiment.

Regarding the experiment shown in Figure 5F of the previous version of the manuscript (now replaced with Figure 5D) in which we showed a comparison of the membrane-bending activities of the p62 variants it is clear that it had some conceptual difficulties. Thus wild type p62 binds very strongly to the membrane via its interaction with LC3B (Figures 2, 3) and the beads via its interaction with ubiquitin (Figure 4). This means that the protein will cover both surfaces and due to the practically irreversible nature of these interactions the interaction of the beads with the membrane are mediated by p62-p62 interactions. In contrast, the oligomerization deficient mutants are less tightly bound to the membrane and the beads and thus are likely to exchange and therefore to be able to mediate simultaneous interaction with LC3B and ubiquitin. We came to the conclusion that we are not comparing the same phenomenon for the different p62 variants. Therefore we have changed the experimental setup by pre-incubating linear tetra-ubiquitin coupled beads with p62 followed by their addition to GUVs coated with LC3B (Figure 5D). This experiment shows that p62 is able to bend the membrane around the beads and that this activity is significantly reduced for the delta PB1 mutant.

3) Figure 6F-Gdo not appear to show significant effects. In 6G in particular, the effects are very small. This is probably due to adaptor redundancy. The authors speculate about other adaptors that might be involved. On balance the data in Figure 6F-G neither support nor oppose the authors' hypothesis.

We agree with the reviewer in that the effects of the oligomerization deficient mutants of p62 on LC3B recruitment to the beads are very small. However, although the effects are indeed very small they are significant. We have left the Figure as is but would be happy to move Figures 6F and 6G to the Figure Supplement or completely from the paper should the reviewers insist.

4) Figure 7C. While the histogram shows what looks like an impressive reduction for the PB1 mutants, I'm confused about how colocalization was scored. By eye it looks like plenty of GFP is colocalized with all of the mutants in Figure 7A.

Figure 7C showed the degree of co-localization of p62 and LC3B at the bacteria. While the degree of LC3B localization to the bacteria was significantly reduced for oligomerization mutants (Figure 7B) the level of co-localization of the p62 and LC3B at the bacteria is mainly influenced by the reduced localization of the p62 oligomerization mutants to the bacteria. We understand that this quantification can be misleading and have moved it to the Figure Supplement ( Figure 7—figure supplement 2.).

5) The advance of the study is to illustrate the enhanced association of p62 with the LC3 and ubiquitin by oligomerization. However, the process is insufficiently characterized. The reviewer suggest calculating a binding curve between the lipid anchored LC3 and different concentrations of different p62 variants. Further, the binding kinetics in addition to the dissociation kinetics indicated in Figure 3 may also be helpful. If oligomerization is just simply increasing the affinity to LC3 and ubiquitin binding, could an engineered p62 with multiple LIFRs fulfill similar purpose?

We thank the reviewer for suggesting these experiments. We have now tested the binding of p62 to LC3 at different concentrations. Instead of GUVs we have used beads since they also allowed us to conduct FRAP experiments without the complication of lateral diffusion of LC3B and the LC3B-p62 complexes on the membrane. Figure 2F, G and Figure 2—figure supplement 3 show the association of wild type p62 as well as the delta PB1 and LIR mutants to LC3B coated beads at different concentrations. Figure 3—figure supplement 2shows the binding of wild type and delta PB1 p62 to LC3B-coated beads overs time.

Figure 3F, G and Figure 3—figure supplement 3. show FRAP analyses of p62 bound to different densities of LC3B on the beads. In combination these FRAP analyses show that the avid binding of p62 to LC3B depends on the density of LC3B on the bead. At high densities the interaction is practically irreversible while at lower densities the effect of oligomerization is lost and the behaviors of oligomeric p62 is like that of the delta PB1 mutant.

Figure 4J shows a FRAP analysis of the interaction of p62 with different densities of ubiquitin on beads. The conclusion of this experiment is that, analogous to the interaction with LC3B, the binding of wild type p62 is more avid to higher ubiquitin densities.

As suggested by the reviewers we have also constructed a p62 version with multiple LC3B interaction site (LIRs). In particular, we have introduced 3 additional LIR motifs next to the endogenous LIR of p62 delta PB1. The FRAP analysis beautifully shows that this 4xLIR mutants binds much more avidly to LC3B coated beads than the single LIR containing protein (Figure 3H and I).

6) Oligomerization decreases the diffusion rate of p62 in the cytosol which may be a negative contributor to its dynamics and efficiency to recognize cargo. Therefore, it is possible that the extent of p62 oligomerization is low but enhanced by cargo binding or association with LC3. Could the author examine the effect of ubiquitin and LC3 on the extent of p62 oligomerization?

We have tested the effect of ubiquitin and LC3B binding on p62 oligomerization. To this end we have employed a pelleting assay as described previously by the Sachse and Johansen labs (Ciuffa et al., Cell Rep. 2015). The assay works well as the presence of p62 in the pellet fraction correlates with ability of p62 to oligomerize (Figures 2B, 2C). In agreement with Ciuffa et al. we found no oligomerization disruption effect by LC3B (Figure 4-figure supplement 1). Also, in agreement with Ciuffa et al. we found that ubiquitin disrupted p62 oligomers to a considerable extent. When we compared different ubiquitin chains for their effect on oligomerization we found that linear, K48-linked and K63-linked chains disrupted p62 oligomers (Figure 4F). Of these K48-linked chains had the strongest disruptive effect. However, oligomeric p62 still binds stronger to ubiquitin chains than to mono-ubiquitin (Figure 4G).

7) The author may consider revising the conclusion in the subsection “"Oligomerization of p62 renders binding to LC3B-coated surfaces irreversible". The authors indicated the "concentrated p62". Here it is difficult to conclude from Figure 3F-H that these soluble LC3 is not concentrated, as once they associate with the oligomerized p62 concentrated on the beads, they may become concentrated too. The reviewer suggests modifying "concentrated" to "membrane associated". Otherwise, it is necessary to quantify the fluorescence intensity of the LC3 on the lipid surface as well as those associated the p62-coated beads to make a comparison. In addition, the author may consider titrating the amount of LC3 on the membrane and quantifying the dissociation rate of oligomerized p62 from the membranes coated with different concentrations of LC3 proteins.

Thank you for pointing out this misleading statement. We now write "Taken together these results suggest that oligomerization of p62 specifically promotes interaction with surface-localized, clustered LC3B by drastically reducing the off-rate of p62 from LC3B-coated surfaces (subsection "Oligomerization of p62 renders binding to LC3B-coated surfaces irreversible, fourth paragraph).

As suggested by the reviewers we have tested the dissociation rate of p62 from beads coated with different densities of LC3B (see response to point 5, Figures 3F, 3G and Figure 3—figure supplement 3. and Figure 3—figure supplement 4).