Comunicación integral de mercadotecnia

to associate with both the trans-Golgi face as well as endosomes (Ho et al., 2006; Liewen et al., 2005; Perez-Victoria et al., 2008; Perez-Victoria et al., 2010) and RNAi depletion resulted in vacuolar/lysosomal mis-sorting (Perez-Victoria et al., 2008; Perez-Victoria et al., 2010). In plants, the complex is known to be required for pollen tube growth, localises to endosomal compartments and is embryo-lethal when knocked-out (Guermonprez et al., 2008; Twell and Pelletier, 2004), although no direct protein sorting studies have been reported on to-date.

1.10 THE DELIVERY OF CARGO TO THE VACUOLE/LYSOSOME

After receptor recycling and the biogenesis of the PVC by ESCRT mediated protein turnover, the final step in the vacuolar sorting pathway is the delivery of the cargo to the vacuole/lysosome. In plants this is facilitated by the maturation of the MVB/PVC into the late-prevacuolar compartment (LPVC). The maturation of the endosomal compartment and the final fusion event to the lysosome are intrinsically linked, requiring the role of the four protein complexes in the process: the Retromer complex, the ESCRT complex, the CORVET complex and the HOPS complex. Although in the literature these processes are often depicted as happening independently they are all sequentially and perhaps causally related to each other, allowing a nascently biosynthesised MVB to fuse to the lysosome.

1.10.1 Late-Prevacuolar Compartment

The Late-Prevacuolar Compartment (LPVC) is the most recently discovered organelle within the secretory system (Contento and Bassham, 2012; De Marcos Lousa et al., 2012) (See Figure 1.1). It was originally identified in 2010 using a recycling defective member of the VSR protein family (Foresti et al., 2010).

1.10 The Delivery of Cargo to the Vacuole/Lysosome 1 INTRODUCTION

It was thus identified as one of the ‘last’ organelles of the secretory pathway before the vacuole. As a recently discovered organelle, the properties are still relatively unknown; it is however, rich with vacuolar cargo and is fusion competent with the vacuole (Bottanelli et al., 2012; Craddock et al., 2008; Foresti et al., 2010), with the fusion observed in electron micrographs (Scheuring et al., 2011). Morphologically, it is similar to the PVC shown by immunogold labelling electron microscopy using antibodies against residents of the LPVC (Haas et al., 2007b). The other likely properties are that it is more acidic than the early secretory pathway, possibly approaching the acidity of the vacuole. This notion is supported by the localisation of the AAA-ATPase Vps4 to the PVC/LPVC (Haas et al., 2007b; Shahriari et al., 2010) which is suggested to acidify the compartment as it matures. This could be potentially due to the presence of a yet unidentified proton transporter. It is also likely to have different membrane properties than the organelles of the early secretory pathway that allow it to become fusion competent with the vacuole, as has been described in the mammalian systems (Huotari and Helenius, 2011).

1.10.2 The Final Maturation of the Endsosome and the Fusion to the Vacuole/Lysosome

The PVC→LPVC maturation event can be considered in some ways a vacuolation of the PVC (See Fig 1.7). In yeast and mammals the process of endosomal fusion is fairly well characterised (Epp et al., 2011). There seems to be a multi-step protein cascade based around the RabGTPases Rab5 and Rab7 as controllers of the process (Rink et al., 2005; Rojas et al., 2008). Both Rab7 and Rab5 have been implicated in binding the HOPS (homotypic vacuolar fusion and protein sorting)/CORVET (class C core vacuole/endosome tethering) protein complexes

1.10 The Delivery of Cargo to the Vacuole/Lysosome 1 INTRODUCTION

(Ostrowicz et al., 2010; Peplowska et al., 2007). HOPS and CORVET are two related multi-protein, endosome associated units (Solinger and Spang, 2013). Both HOPS and CORVET share the Vps11/16/18/33 tetramer, which interacts with Vps39 and Vps41 in the HOPS complex and Vps8 and Vps3 in the CORVET complex (Epp et al., 2011).

Current theories suggest that the CORVET complex is involved at an earlier stage of endosome maturation/fusion as mutants show abnormal endosome morphology lacking ILVs (Peplowska et al., 2007). The CORVET complex has also been shown to interact with the Rab5GTPase, as part of the maturation process. Whereas the Rab5/CORVET complex is thought of as essential for endosome maturation, the Rab7/HOPS complex has been implicated in the fusion process (Stroupe et al., 2009).

Some reports suggest that the loss of Rab5 from the endosome is coupled by the accumulation of Rab7. This CORVET to HOPS cascade seems to be mediated by Rab7 GEF Mon1-Ccz1, that is recruited by the CORVET complex and in turn recruits Rab7. During this transition there also seems to be simultaneous recruitment of the retromer complex by the Rab7 (Balderhaar et al., 2010). The Mon1-Ccz1 negotiated binding and nucleotide exchange of Rab7 causes Vps8 and Vps3 to dissociate from the CORVET complex allowing its transition into the HOPS complex by recruitment of Vps39 and Vps41.

Work with homotypic fusion in yeast has comprehensively described the events that allow membrane fusion to the vacuole (Wickner, 2010). Interestingly, homotypic fusion seems to be the same as endosomal fusion from a molecular perspective, further supporting the idea that endosome maturation is analogous to vacuolation. In a process that has been completely reconstituted in vitro, the fusion event itself

1.10 The Delivery of Cargo to the Vacuole/Lysosome 1 INTRODUCTION PVC LPVC Vacuole m Retromer recycling CORVET complex HOPS complex Rab5 Rab7 ESCRT complex

Figure 1.7: Maturation of the Endosomes

is very similar to a SNARE arbitrated fusion event, which is supplemented by the action of Rab7 and the HOPS complex. There are two SNARE complexes implicated in the process; the first complex consists of the three Q-SNAREs Vam3, Vam7 and Vti1 which can interact with the second complex containing the R-SNARE Nyv1 or the R-SNARE Ykt6 (Mima et al., 2008; Thorngren et al., 2004). The tethering of the vacuolar SNARE complex to the endosome seems to be through interaction with the HOPS complex, assisted by Rab7. Through an unknown mechanism, perhaps the hydrolysis of the Rab7 followed by the canonical SNARE activity, the endosome is brought into close contact with the vacuole coupled with membrane fusion.

A similar process has been partially documented in plants. Rab7 has been shown to partition to the vacuole, and Rab5 to the LPVC (i.e. late endosome) (Bottanelli et al., 2012; Foresti et al., 2010). Furthermore, dominant negative (asparagine to isoleucine, NI) mutants of both Rab5 and Rab7 prevented the fusion of the LPVC to the vacuole (Bottanelli et al., 2012). Finally, it was observed that the Rab5NI seemed to stop the maturation of the LPVC, as both PVC markers and LPVC markers colocalised. This is in contrast to the Rab7NI that seemed to preserve