Simplified overview of p97 mutation and dose dependent effects.

<p>Simplified overview of p97 mutation and dose dependent effects.</p

Cell growth in shaking culture and on <i>Klebsiella aerogenes</i> are altered in mutant strains expressing p97-RFP or p97^R155C-RFP.

<p>(A) Strains expressing p97-RFP or p97^R155C-RFP display specific growth defects in shaking culture. Please note the logarithmic scale of the y-axis. (B) Growth on <i>K. aerogenes</i> lawns. Mutation specific and dose dependent effects are seen in both wild-type and ATG9^KO strains. Growth of AX2 on day 5 was set to 100%.</p

Predicted and observed experimental outcomes of the expression of p97 and p97^R155C in AX2 wild-type and ATG9^KO cells.

<p>The table summarizes the predicted changes based on the model of p97 and ATG9 interaction and mutual inhibition as illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046879#pone-0046879-g009" target="_blank">Fig. 9</a>. Experimental results in black; model validity in green; “no” and “no” with uppercase “1)”, see Discussion section; “ref value”, changes were separately compared to wild-type and ATG9^KO backgrounds; ↑, ↓, →, increase, decrease or no change with respect to the reference value. Inverse, inverted mutation specific effect. Rescue, partial or full rescue of the ATG9^KO phenotype.</p

Subcellular localization of p97 and its co-localization with ubiquitin in wild-type and mutant strains.

<p>(<b>A</b>) Visualization of subcellular localization of p97 in AX2 wild-type cells with polyclonal antibodies p97_8_6841 directed against amino acids 23–73 and p97_9_6574 directed against amino acids 254–310. (<b>B</b>) Subcellular localization of p97 (<b>left panel</b>) using the p97_8_6841 polyclonal antibody and ubiquitin (<b>middle panel</b>) using the P4D1 monoclonal antibody (NEB, Germany) in AX2 wild-type cells and mutant strains. Merged images and DAPI staining to visualize nuclei (<b>right panel</b>). <b>Upper row</b>: AX2 wild-type cells; <b>middle row</b>: ATG9^KO mutant; <b>bottom row</b>: ATG9^KO/p97^R155C-RFP double mutant. Please note that ubiquitin positive protein aggregates frequently co-localize with p97 in the ATG9^KO mutant (arrows) but not in the ATG9^KO/p97^R155C-RFP double mutant (double arrowheads). The ATG9^KO mutant also contains protein aggregates that are either positive for p97 (arrowhead) or ubiquitin (double arrowhead). Cells were fixed with cold methanol and stained with the indicated antibodies. Scale bars are 10 µm and 2 µm in inset.</p

Sequence identity and sequence similarity of p97 from different organisms.

<p>Sequence identity, left, and sequence similarity, right, was determined by aligning the corresponding protein sequences using BLAST align program (bl2seq) at the NCBI. Percentage values are given. Hs: <i>Homo sapiens</i>, <i>Mm: Mus musculus, Rn: Rattus norwegicus, Xl: Xenopus laevis, Sc: S. cerevisiae, Dd: D. discoideum.</i></p

p97 and p97^R155C form heteromers in vivo.

<p>(<b>A</b>) Coomassie stained SDS-PAGE gel of a p97 co-immunoprecipitation experiment. Input: soluble cell lysate of either AX2/p97^R155C-RFP (<b>left column</b>) or AX2 (<b>right column</b>) cells; Control (ctrl): beads incubated with bovine serum albumin instead of the polyclonal RFP antibody; CoIP: co-precipitated proteins by the RFP antibody. (<b>B</b>) Immunoblot verification of the presence of p97 and p97^R155C-RFP in the immunoprecipitate and input. (<b>C</b>) Immunoblot verification of the presence of p97^R155C-RFP.</p

Interaction and mutual inhibition of p97 and ATG9.

<p>(<b>A</b>) The analysis of phototactic ability illustrates mutual inhibition of p97 and ATG9 in conjunction with inhibition of this process in AX2 cells. (<b>B</b>) Expression of p97^R155C in AX2 wild-type cells frees ATG9 to exert its inhibitory activity. (<b>C</b>) Expression of wild-type p97 in wild-type AX2 cells results in slightly reduced phototaxis which can be explained by a dose dependent effect. (<b>D</b>) In the absence of ATG9, its inhibitory activity versus p97 is lost leaving p97 free to strongly inhibit phototaxis. (<b>E</b>) Partial rescue of phototaxis in the ATG9^KO/p97^R155C-RFP double mutant. (<b>F</b>) Since phototaxis is already lost in ATG9^KO cells, the expression of p97-RFP cannot lead to a further impairment. Line width correlates with the strength of the inhibitory effect. Dotted line, residual activity. Please refer to the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046879#s4" target="_blank">Discussion</a> section for further details on this model of interaction and mutual inhibition.</p

Expression of p97^R155C-RFP rescues fruiting body formation in ATG9^KO cells.

<p>Neither expression of p97-RFP nor p97^R115C-RFP changed fruiting body formation in AX2 wild-type cells (<b>left column</b>). In the ATG9^KO strain fruiting body formation is completely lost (<b>top right image</b>). While expression of p97^R155C-RFP in the ATG9^KO strain fully rescued the fruiting body formation (<b>middle right image</b>), no obvious rescue of fruiting body formation was observed upon expression of p97-RFP (<b>bottom right image</b>). The assay was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046879#pone.0046879-Tung1" target="_blank">[28]</a>.</p

Expression of p97^R155C-RFP impairs phototaxis in AX2 cells and rescues the phototaxis defect of ATG9^KO cells.

<p>The ability of AX2 wild-type and of mutant slugs to migrate towards a light source (wavy line) was tested. While AX2 slugs (<b>top left image</b>) and AX2 slugs expressing p97-RFP (<b>bottom left image</b>) nicely migrated towards the light source, phototactic ability was severely impaired in the AX2/p97^R115C-RFP strain (<b>middle left image</b>). In the ATG9^KO strain phototaxis is completely lost (<b>top right image</b>). While expression of p97^R155C-RFP in the ATG9^KO strain partially rescued the phototactic ability (<b>middle right image</b>), no rescue of phototactic ability was observed upon expression of p97-RFP (<b>bottom right image</b>). The phototaxis assay was performed as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046879#pone.0046879-Tung1" target="_blank">[28]</a>.</p