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leaf epidermal cells (Paris et al., 1996). This was later refuted with convincing evidence that the defining markers of the storage/vacuolar organelles, members of the aquaporin family, co-localised in the leaf epidermal tissue (Hunter et al., 2007). Nevertheless, the presence of the storage vacuole in seed tissue is uncontested and can be considered a unique adaptation to the lytic compartment of the vacuole. Post-Golgi transport in eukaryotic cells is a process that relies heavily on active sorting motifs and sorting receptors.

Due to the involvement of multiple organelles, the protein transport pathways to the vacuole are complex and controversial. This is more-so in the plant model system where there are contrasting views on the role of the trans-Golgi network, and the proposed presence of multiple vacuoles. As a result there are several pertinent questions on this topic. A selection of these questions are addressed in this thesis and therefore the various models of vacuolar protein sorting are explored in detail below.

1.7 VACUOLAR SORTING SIGNALS AND RECEPTORS

To divert proteins from the default pathway to the cell surface, specific sorting signals are needed to interact with a membrane spanning receptor, analogous to ERD2 described above. This indirectly allows the soluble cargo to interact with the vesicle coat protein apparatus that is in the cytosol. Vacuolar sorting motifs on the soluble cargo are comprised of protein surface structures in yeasts and plants. In plants, there are C, N and internal sorting sequences characterised for a variety of cargo molecules (Matsuoka and Neuhaus, 1999). In yeast there are at least two different consensus cargo sorting signals; one type present in carboxypeptidase Y (CPY) and vacuolar aspartyl protease proteinase A and another present in vacuolar

1.7 Vacuolar Sorting Signals and Receptors 1 INTRODUCTION

Figure 1.5: The Cross-Kingdom Lysosomal Sorting Determinants

aspartyl protease proteinase B (Robinson et al., 1988). Finally, in mammalian cells there are also at least two different types, one protein motif and a unique phosphomannyl post-translational motif (Braulke and Bonifacino, 2009; Kornfeld, 1992).

There are a number of characterised receptor molecules for vacuolar sorting which all share common topology of a type-I-membrane protein (Figure 1.5). It is possible that many vacuolar sorting receptors share a common ancestral form. The focus for this thesis is the model plant Vacuolar Sorting Receptor (VSR), described below (Section 1.7.3), however there are analogous receptors in other systems that have contributed to the understanding of receptor-mediated sorting in general which will be also described in detail.

1.7.1 The Mammalian Sorting Receptors

The first identified and characterised lysosomal sorting receptor was the mammalian mannose-6-phosphate receptor (M6PR) which has been shown to have a direct role in lysosomal sorting as well as endocytosis (Kornfeld, 1992). The M6PR was shown to bind to phosphomannosyl residues which are conjugated to

1.7 Vacuolar Sorting Signals and Receptors 1 INTRODUCTION

proteins in the Golgi apparatus (Kaplan et al., 1977), allowing for entry into CCVs (Campbell and Rome, 1983). The original observations identified the M6PR as an endocytic determinant, however, it was quickly realised that an anterograde sorting step would involve the M6PR and clathrin during biosynthesis of the receptor (Pearse and Bretscher, 1981; Rothman and Fine, 1980). This hypothesis was confirmed when it was shown that the M6PR is concentrated in the Golgi apparatus (Brown and Farquhar, 1984). It was also shown that there are two types of M6PR, one, a small ‘cation-independent’ (CDM6PR or MPR46) and the other much larger ‘cation-dependent’ receptor (CIM6PR or MPR300) (Hoflack and Kornfeld, 1985).

The mannose-6-phosphate receptors represent a unique lysosomal sorting system as they do not recognise cargo by a protein-protein interaction, but by a phosphomannosyl residue covalently attached to proteins post-translationally. Other mammalian sorting receptors use protein-protein interactions to mediate sorting, for example sortilin (Nielsen et al., 2001). Sortilin is a type I membrane spanning receptor with a short C-terminus that indirectly interacts with the clathrin protein coat. The lumenal domain of sortilin interacts with and traffics sphingolipid activator proteins, a family of molecules essential for glycosphingolipid catabolism in the lysosome (Lefrancois et al., 2003).

1.7.2 The Fungal Vps10p

In yeast, the best characterised sorting receptor is the sortilin homologue Vps10p. Vps10p was originally proposed to be involved with vacuolar protein sorting (vps) when a vps10 mutant was highlighted in a genetic screen for mutant yeast strains that secreted the vacuolar model cargo carboxypeptidase Y (CPY) (Raymond

1.7 Vacuolar Sorting Signals and Receptors 1 INTRODUCTION

et al., 1992; Robinson et al., 1988). Chemical cross-linking of Vps10p and its ligand indicated that Vps10p was the sorting receptor for the vacuolar hydrolase CPY (Marcusson et al., 1994). The stoichiometry of this interaction was shown to be approximately 1:1 (Cooper and Stevens, 1996). CPY is proteolytically processed when it arrives at the vacuole, which abolishes the interaction with Vps10p and serves a routine tool to monitor CPY anterograde transport in sorting assays and mutant screens (Cooper and Stevens, 1996).

1.7.3 The Plant Vacuolar Sorting Receptor

The model receptor used in this thesis is the plant vacuolar sorting receptor (VSR). There are 7 VSRs in A. thaliana, two of which are highly homologous direct repeats (De Marcos Lousa et al., 2012; Hadlington and Denecke, 2000). Using a biochemical approach the VSR family of proteins was originally identified in 1994 (Kirsch et al., 1994). In this landmark study, the plant vacuolar sorting receptor was shown to interact with peptide motifs of the vacuolar sorted protein aleurain. Subsequently, it was shown that VSRs have a large lumenal domain, which mediates interaction with the cargo, and a short cytosolic C-terminus that mediates interaction with the cytosolic apparatus (De Marcos Lousa et al., 2012). The lumenal domain has been studied with respect to ligand binding, and a variety of cargo molecules have been identified that interact with the receptor for their delivery into the vacuole. Specific binding of a recombinant VSR, lacking its TM domain and cytosolic tail showed that a monomeric lumenal domain alone can interact with the cargo (Cao et al., 2000; Sanderfoot et al., 1998). Surface plasmon resonance using purified lumenal VSR domains expressed in insect cells confirmed this (Watanabe et al., 2002) and was further supported by secreted soluble VSR domains purified from the culture medium of tobacco Bright Yellow 2 (BY2)