In order to understand the molecular mechanism of ESCRT-mediated protein sorting in the context of endomembrane system, we aimed at characterizing the plant equivalent of the complex that resembles the heart of the ESCRT machinery in yeasts and mammals: ESCRT-II. In this regard, we aimed at characterizing the interactions between its putative subunits Vacuolar Protein Sorting 22 (VPS22), VPS25 and VPS36, as well as the respective localization of those three proteins.
For the localization studies in planta, we first generated Arabidopsis lines homozygous for a pVPS36::VPS36-GFP construct and the vps36 knock-out causing SALK_130246.49.85.x insertion. Expression of VPS36-GFP in these lines rescued the otherwise lethal phenotype of the vps36 knock-out. Confocal analysis of rescued plants revealed a strong presence of VPS36-GFP at the PM. GFP signals were also observed less frequently at dot-like structures, There they partially co-localized with the styryl Fei-Mao (FM) dye 4-64. As this dye is an endocytic tracer, this indicated that plant VPS36, like its counterpart in yeast, partially localizes at endosomal structures (Babst et al., 2002b; Bolte et al., 2004). Even though VPS36-GFP was able to rescue the vps36 knock-out phenotype and thus had to be at least partially functional, its PM localization was rather unexpected and might have been caused by the presence of the GFP-tag. To rule this out and to monitor for the intracellular distribution of ESCRT- II in general on an ultrastructural level, we aimed at localizing all three putative subunits, namely VPS22, VPS25 and VPS36, in wildtype plants by immune-electron
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microscopy. For this, I raised specific antibodies against each of these three proteins and characterized them in respect to their ability to bind denatured and native VPS22, VPS25 or VPS36, respectively. Since all of them were proven to cross react with native antigens in immune precipitation assays, we employed them for in-situ studies on high- pressure frozen and freeze substituted Arabidopsis roots. To our great surprise, all three putative ESCRT-II subunits were found to be most prominently localized at the PM and only to a lesser extend at the TGN and the MVB. We interpreted the fact that all three proteins showed a similar distribution within the cell, including the intriguing and plant-specific accumulation at the PM, as a first indication that they actually form a complex. Likewise, transiently co-expressed VPS36-GFP, VPS22-RFP and VPS25- BFP2 exhibited perfect co-localization at the PM of tobacco mesophyll protoplasts. Furthermore, the protoplast system allowed for individual expression of fluorescently tagged VPS22, VPS25 or VPS36, to study their localization independently from each other. Under these circumstances, only VPS36 fusions were recruited to the plasma membrane. In contrast, VPS22-RFP and VPS25-RFP were completely cytosolic and showed no co-localization with a co-expressed PM marker. Since endogenous VPS22 and VPS25 had been detected at the PM in our ultrastructural analysis and VPS22- and VPS25-fusion proteins had been observed at the PM when co-expressed with VPS36-GFP, I hypothesized that their localization depends on the availability of VPS36. To test for this hypothesis, either VPS22-RFP or VPS25-RFP was co- expressed with VPS36-GFP. Now, both of them co-localized with VPS36-GFP at the PM indicating that VPS36 attaches to the PM independently of VPS22 and VPS25 and also mediates the recruitment of the latter two to this very site.
7.3.2 VPS22, VPS25 and VPS36 interact in vivo
Even though VPS22, VPS25 and VPS36 were found to co-localize and PM localization of all three seemed to depend on the same VPS36-mediated mechanism, it remained unclear, whether they form a complex in plant cells. Proofing the existence of such a complex required the demonstration of the interactions between its putative subunits. To perform interaction analyses in vivo, I opted for a FRET-FLIM approach. Here, VPS36-GFP was employed as energy donor, while either VPS25-RFP or VPS22-RFP was co-expressed as potential acceptors. Both RFP-fusion proteins, in sharp contrast to Cytosolic RFP (cytRFP), reduced the fluorescent lifetime of VPS36-GFP in a highly
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significant fashion. This indicated a distance of less than 10 nm between the acceptor- and the donor-fluorophores implying an interaction between VPS36 and VPS22 as well as VPS25.
These findings hinted towards VPS36 being the central protein of plant ESCRT-II as it interacted with both other subunits and mediated their membrane attachment. To further characterize the ESCRT-II complex formation, we generated truncated mutants and tested for their ability to interact with VPS22 and VPS25. Already the deletion of 33 amino acids at the C-terminus of VPS36 (VPS36Δ33) abolished its capability of binding VPS25. However, VPS22-RFP was still able to bind to VPS36Δ33-GFP. This indicated that the VPS22-interaction site within VPS36 is located closer to the N- terminus than the VPS25-interaction site and showed that the loss of the ability to bind VPS25 was indeed caused by the truncation of an interaction motif and not due to general misfolding of VPS36Δ33-GFP. To also map the VPS22-interaction site, we further removed 33 amino acid stretches from the C-terminus of VPS36. While VPS36Δ66 and VPS36Δ99 still bound VPS22-RFP, VPS36Δ132-GFP was no longer able to do so. Because VPS36Δ132-GFP was still recruited to the PM, gross misfolding of this protein could again be excluded as a cause for non-interaction. The occurring FRET-effect in the presence of RFP-NbG additionally demonstrated the
functionality of this molecule as an energy donor. These results allowed the conclusion that the VPS22-interaction motif is situated between the 132th and 99th most C- terminal residues of VPS36.
After characterizing the interaction of VPS22 and VPS25 with VPS36, I intended to probe the putative VPS22-VPS25 interaction, which has been suggested based on yeast-2-hybrid screening results (Richardson et al., 2011; Shahriari et al., 2011). For this, VPS25-GFP was co-expressed with VPS22-RFP, VPS36-RFP as positive control and cytRFP as negative control. VPS22-RFP and VPS36-RFP but not cytRFP reduced the fluorescent lifetime of VPS25-GFP in FRET-FLIM studies significantly. This clearly indicated that VPS22 and VPS25 interact in plants but also raised the question whether VPS22 and VPS36 bind to the very same VPS25 molecule or to two different VPS25 moieties.
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