<i>In</i><i>vitro</i> production of HOPS and CORVET subunits and pull down analysis of the subunit interactions.

<p>(A) Schematic representation of the experimental design. (B) SDS-PAGE analysis of the LTE lysate samples expressing individual subunits of the HOPS/CORVET complex. The samples were spiked with BODIPY-Lys-tRNA and the reaction products were visualized by scanning the unstained SDS-PAGE gels. (C) Interactions of Vps11 with other subunits of HOPS/CORVET complex. The GFP-tagged Vps11 was co-expressed with other subunits under conditions described in (B) and GFP-tagged proteins were isolated on the anti-GFP beads, eluted with SDS-PAGE loading buffer and resolved on the denaturing SDS-PAGE. The proteins were visualized by fluorescence scanning. (D) Interaction map of HOPS complex subunits based on the current study (red lines) and literature data (black and green lines). (E) the map of interactions in CORVET complex annotated as in D. </p

Analysis of HOPS/CORVET complex assembly domains (A) sequence alignment of VPS11 interacting domains of Vps3 and Vps16.

<p>(B) Multiple sequence analysis of the complex assembly domains of Vps3, Vps16 and Vps39. (C) Sequence alignment of the Vps8 and Vps41 interacting domains. (D) Reconstitution of the interaction core of HOPS complex. The truncated subunits we co-expressed with GFP-tagged Vps41 subunit and isolated on the anti-GFP matrix as described above. (E) Organization of HOPS complex based on the low resolution structure of yeast produced complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B13" target="_blank">13</a>]. (F) Proposed mode of complex assembly based of the EM reconstruction and the presented biochemical results.</p

Recombinant HOPS can support vacuole fusion.

<p>Vacuoles were purified from each of the two tester strain (<i>pep4</i>∆ and <i>pho8</i>∆) that each carry the <i>vps11-1</i> mutation (Stroupe [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B29" target="_blank">29</a>]and Wickner, 2006). Fusion reactions (30 µl total) contained 3 µg of each vacuole type in fusion salt (0.5 mM MgCl₂, 125 mM KCl, 20 mM PIPES/KOH, pH 6.8, 200 mM sorbitol) and an ATP-regenerating system [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B14" target="_blank">14</a>]. (A) The indicated volumes of each fraction of the HOPS purification were titrated into the fusion reaction. Reactions were incubated for 90 min at 26°C, and then assayed for Pho8 activity by addition of p-Nitrophenolphosphate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B14" target="_blank">14</a>]. As a control, purified yeast HOPS complex [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081534#B16" target="_blank">16</a>] was added at the indicated concentrations. (B) Titration of fraction 9 at higher volumes into the fusion reaction. The analysis was conducted as in (A). Control indicates the addition of volumes of purification buffer into the reaction. </p

<i>In</i><i>vitro</i> assembly of subcomplexes of HOPS and CORVET complexes.

<p>(A) An example of subcomplex containing Vps11, Vps16, GFP-Vps18 and Vps33 co-expressed in LTE extract and isolated on the anti-GFP matrix. (B) Graphic summary of subcomplexes isolated in this study. (C) Fluorescence scan of the SDS-PAGE gel loaded with <i>in </i><i>vitro</i> reconstituted and affinity purified HOPS complex. The positions of the individual subunits are indicated on the right hand side. (D) Coomassie stained SDS-PAGE loaded with the HOPS containing fraction eluted from the gel filtration column. (E) Identification of the complex forming domains in the Vps subunits of HOPS and CORVET complex. GFP-tagged Vps11 was co-expressed with the truncated variants of Vps39 subunit. The samples were processed as in A. </p