<i>In-vitro</i> cleavage characteristics of xAtg4B^14-384.

<p><b>A,</b> Time course. Substrates (100 μM) were incubated at 0°C with 500 nM of xAtg4B^14-384. At indicated time points, aliquots were withdrawn. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are full-length substrate proteins (fl) and the C-terminal cleavage products (ccp). For a side-by side comparison of selected protease fragments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s004" target="_blank">S4 Fig</a>. <b>B</b>, Temperature dependence of substrate cleavage. 100 μM of xLC3B-MBP (left) or xGATE16-MBP (right) were incubated with xAtg4B^14-384 for 1 h at defined temperatures. Note that in comparison to the xGATE16-MBP substrate, twice as much protease was used for cleavage of the xLC3B-MBP substrate. For a comparison of selected protease fragments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s008" target="_blank">S8 Fig</a>. <b>C,</b> Salt sensitivity. 100μM of each substrate was incubated for one hour at 0°C with 500 nM xAtg4B^14-384 at NaCl concentrations ranging from 0.2 to 1.5 M. For a comparison of selected protease fragments see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s008" target="_blank">S8 Fig</a>. <b>D,</b> P₁' preference. Protease substrates used to analyze the P₁' preference of xAtg4B^14-384 followed the general outline shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.g002" target="_blank">Fig 2A</a>. Here, however, the P₁' position of the P₁-P₁' scissile bond had been mutated to the potentially non-preferred residues methionine (Met), tyrosine (Tyr), arginine (Arg), glutamic acid (Glu), or proline (Pro). Solution cleavage assays were performed with indicated concentrations of xAtg4B^14-384 for 1 h at 0°C. Bands marked with an asterisk (*) refer to the protease.</p

<i>In-vitro</i> substrate cleavage by xAtg4B fragments.

<p><b>A</b>, Schematic representation of the protease substrates xLC3B-MBP (top) and xGATE16-MBP (bottom). Both fusion proteins contain an N-terminal polyHis-tag, a protease recognition site (xLC3B or xGATE16) and MBP (<i>E</i>. <i>coli</i> maltose-binding protein, MBP) as a model target protein. To ensure a comparable accessibility, the scissile bond is followed by the identical tri-peptide (AGT; Ala-Gly-Thr) in both substrate proteins. For simplicity, substrate names do not contain the polyHis-tag. <b>B</b>, <i>In-vitro</i> cleavage assay. 100 μM substrate (xLC3B-MBP (left) or xGATE16-MBP (right)) was incubated for 20 h at 37°C with defined concentrations of the indicated xAtg4B protease fragments. Cleavage products were separated by SDS-PAGE and stained with Coomassie G250. Shown are full-length substrate proteins (fl) and the C-terminal cleavage products (ccp). To estimate the completeness of cleavage, the band intensities were compared to a cleavage standard (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s003" target="_blank">S3 Fig</a>). <b>C</b>, Activity of xAtg4B fragments at 0°C. Indicated concentrations of xAtg4B protease fragments were incubated for 1 h at 0°C with 100 μM of the substrates sketched in <b>A.</b> For a similar comparison of xAtg4B fragments at 25°C see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s004" target="_blank">S4 Fig</a> For examples of complete gels see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s005" target="_blank">S5 Fig</a>.</p

Thermal stability of xAtg4B fragments.

<p><b>A,</b> Long-term thermal stability. Indicated xAtg4B fragments were pre-incubated for 16 h at indicated temperatures in the presence of 20 mM DTT under argon to protect the active site cysteines from oxidation. The remaining activity was then assayed by treating 100 μM of xLC3B-MBP or xGATE16-MBP substrate with each protease fragment for 1 h at 0°C. <b>B,</b> Dynamic light scattering (DLS) analysis. xAtg4B fragments were diluted to a final concentration of 10 μM and assayed by DLS. The temperature was automatically raised by 1°C every 10 min. DLS signals were acquired just before each temperature step.</p

<i>In-vitro</i> cross-reactivity with other tag-cleaving proteases.

<p><b>A</b>, Schematic representation of substrates used for (B) and (C). The TEV protease substrate contains an N-terminal His₁₀-ZZ tag preceding the TEV protease recognition site. All other substrates follow the scheme described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.g002" target="_blank">Fig 2A</a>, the protease recognition site, however, is replaced by the respective ubiquitin-like protein (UBL). <b>B</b>, Cross-reactivity between recombinant tag-cleaving proteases. bd, Brachypodium distachyon; tr, <i>Triticum aestivum</i> (summer wheat); xUb, <i>Xenopus</i> ubiquitin. Bands marked with an asterisk (*) originate from the respective protease. For complete gels see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s006" target="_blank">S6 Fig</a>. <b>C</b>, Detailed titration analysis of cross-reactivity between <i>Xenopus laevis</i> (x), <i>S</i>. <i>cerevisiae</i> (sc) and wheat (tr) Atg4 homologs.</p

Cleavage efficiency at limiting substrate concentrations.

<p>The concentration of indicated protease fragments and the substrates xLC3B-MBP (left) or xGATE16-MBP (right) was titrated at constant protease/substrate ratio (1:1000 or 1:2000, respectively). After cleavage (1 h at 0°C), a fraction of each reaction corresponding to 1.2 μg (≈20 pmol) of substrate protein was analyzed by SDS-PAGE. Due to the different substrate concentrations, the absolute volume of the cleavage reaction analyzed by SDS-PAGE had to be adjusted accordingly.</p

On-column cleavage using xAtg4B^14-384.

<p><b>A</b>, Schematic representation of substrate proteins used in (<b>B</b>)—(<b>E</b>). The N-terminal domain of <i>E</i>. <i>coli</i> IF2 (IF2d1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.ref065" target="_blank">65</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.ref066" target="_blank">66</a>]) serves as a spacer. <b>B and C</b>, A silica-based Ni²⁺ chelate resin was pre-loaded with similar amounts of His₁₄-bdNEDD8-mCherry and either His₁₄-IF2d1-xLC3B-GFP (<b>B</b>) or His₁₄-IF2d1-xGATE16-GFP (<b>C</b>). 50 μl aliquots were treated with indicated concentrations xAtg4B^14-384 for 1 h at 4°C. Control incubations were performed with 4 μM bdNEDP1 or with buffer containing 400 mM imidazole. Resins and eluates were photographed while illuminated at 366 nm. GFP and mCherry in the eluate fractions were quantified via their specific absorptions. Quantification results are given below the respective eluate fractions. <b>D and E</b>, Protein purification using on-column cleavage by xAtg4B^14-384. Indicated substrates were over-expressed in <i>E</i>. <i>coli</i>. After lysis and ultracentrifugation, the soluble material was incubated with a Ni²⁺ chelate resin. The resin was washed and treated with 500 nM xAtg4B^14-384 at 4°C. At indicated time points, the concentration and purity of the released MBP was determined using the calculated absorption coefficient at 280 nm (OD₂₈₀) and SDS-PAGE, respectively. Proteins remaining on the resin after 60 min were eluted by 500 mM imidazole. The time course of elution is shown in (<b>D</b>), the OD₂₈₀ reading at 60 min elution time was set to 100%. Relevant steps of the purifications are shown in (<b>E</b>).</p

Stability of UBL fusions in eukaryotic lysates and in <i>S</i>. <i>cerevisiae</i>.

<p><b>A</b>, Schematic representation of substrates used for (<b>B</b>). <b>B</b>, Stability of protease substrates in cell extracts. Note that in wheat germ extract no proteolytic fragments originating from SUMOstar-, xLC3B- or xGATE16-containing substrates can be detected. For complete blots and stained membranes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s010" target="_blank">S10 Fig</a>. <b>C</b>, Schematic representation of substrates used for expression in <i>S</i>. <i>cerevisiae</i> (<b>D</b>) harboring an N-terminal ZZ-tag, an ubiquitin-like protein (UBL) and a C-terminal Citrine. <b>D</b>, <i>In-vivo</i> stability of protease substrates in <i>S</i>. <i>cerevisiae</i>. Indicated protease substrates were over-expressed in a <i>S</i>. <i>cerevisiae</i> strain constitutively expressing H2B-CFP. Total cell lysates were analyzed by Western blot with antibodies recognizing the ZZ-tag (upper panel) or Citrine and CFP (middle panel). Equal loading was confirmed by staining the membrane after blotting (lower panel). Bands marked with an asterisk (*) originate from ZZ-tagged proteins cross-reacting with the anti-Citrine/CFP antibody. For complete original blots and stained membranes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125099#pone.0125099.s011" target="_blank">S11 Fig</a>. <b>E</b>, Cleavage of UBL substrates in extracts and in <i>S</i>. <i>cerevisiae</i>. ++, highly efficient cleavage; +, cleavage;–, traces cleaved;––, no cleavage; n.d.: not determined; ¹, data not shown.</p

One-step protein purification from <i>S</i>. <i>cerevisiae</i>.

<p>ZZ-UBL-Citrine fusions sketched in (<b>A</b>) were over-expressed in <i>S</i>. <i>cerevisiae</i>. Cells were lysed and the soluble material was incubated with an anti-ZZ affinity resin. After washing off unbound material, highly pure Citrine was eluted by treatment with 0.1 μM SUMOstar protease (<b>B</b>), 1 μM xAtg4B^14-384 (<b>C</b>) or 1 μM bdNEDD8 (<b>D</b>) for 1 h at 4°C. Material remaining on the resin was analyzed after elution with SDS sample buffer. The asterisk (*) denotes the full-length xLC3B fusion protein. The filled circle (•) marks band partially corresponding to low levels of free Citrine originating from <i>in-vivo</i> cleavage of the respective SUMOstar and bdNEDD8 fusion proteins.</p

Myelin Membrane Assembly Is Driven by a Phase Transition of Myelin Basic Proteins Into a Cohesive Protein Meshwork

<div><p>Rapid conduction of nerve impulses requires coating of axons by myelin. To function as an electrical insulator, myelin is generated as a tightly packed, lipid-rich multilayered membrane sheath. Knowledge about the mechanisms that govern myelin membrane biogenesis is required to understand myelin disassembly as it occurs in diseases such as multiple sclerosis. Here, we show that myelin basic protein drives myelin biogenesis using weak forces arising from its inherent capacity to phase separate. The association of myelin basic protein molecules to the inner leaflet of the membrane bilayer induces a phase transition into a cohesive mesh-like protein network. The formation of this protein network shares features with amyloid fibril formation. The process is driven by phenylalanine-mediated hydrophobic and amyloid-like interactions that provide the molecular basis for protein extrusion and myelin membrane zippering. These findings uncover a physicochemical mechanism of how a cytosolic protein regulates the morphology of a complex membrane architecture. These results provide a key mechanism in myelin membrane biogenesis with implications for disabling demyelinating diseases of the central nervous system.</p> </div

β-sheet structure mediates amyloid-like self-assembly of MBP.

<p>(A) Secondary structure determination from FTIR spectroscopy of wild-type MBP in the presence of 20 mM NaOH. Bars represent range from two independent experiments. (B) Secondary structure determination of wild-type and the F→S mutant after addition to the SLBs with the inner myelin leaflet lipid composition. (C) Aggregation-prone stretches within MBP sequence. (D) Transmission electron microscopy of peptide 1 and peptide 2 (left and middle panel) incubated in 25 mM HEPES (pH 7.5), 150 mM KCl, and 0.5 mM MgCl₂ for several days. The lower panel shows the fibrillar aggregates obtained in 20 mM HCl, 500 mM Na₂SO₄, and 5% ethanol for wild-type MBP, but not the MBP F→S mutant. (E) Thioflavin S staining of P18 MBP deficient <i>shiverer</i> and wild-type mice brain. Quantification of relative fluorescent intensity in corpus collosum. Bars show mean ± SEM (<i>n</i> = 3 animals, **<i>p</i><0.01, <i>t</i> test).</p