Clathrin-mediated endocytosis is an important vesicle biogenesis pathway. It is the chief means for internalization of transmembrane proteins, bound cargos and lipids from the plasma membrane and may also occur in the trans-golgi network and on some endosomes (Bonifacino & Lippincott-Schwartz, 2003). The primary function of CME is nutrient and growth factor uptake into the cell; however, despite its specificity and tight regulation, this process is susceptible to hijacking by toxins and viruses. CME commences with the formation of clathrin coated pits (CCP) from which cargos are packed into vesicles that are surrounded by a coat predominantly made of clathrin and adaptor proteins (Benmerah & Lamaze, 2007). Concurrently, a number of endocytic accessory proteins and alternative adaptors are recruited to the cell surface (Fig. 1). The accessory proteins can simultaneously or sequentially become engaged in interactions with other components of this endocytic pathway, such as lipids and transmembrane proteins anchored in plasma membranes. This series of inter-connecting events has recently been described in several organisms as a dynamic network in which adaptor protein 2 complex (AP2) and clathrin hold a central position (Schmid et al., 2006; Schmid & McMahon, 2007). While accessory proteins primarily play a regulatory role in determining the contents and initiating the formation of coated pits, AP2 and clathrin maintain additional functions as structural components of coated vesicles. Forty-five endocytic genes were annotated in the A. pisum genome (Table 1). This finding represents a quasi-perfect correlation with D. melanogaster (Table S1), the only difference identified between these two species is the absence of the ced-6 gene in the A. pisum genome but the additional presence of the low-density lipoprotein receptor adaptor protein 1 gene (discussed below). This result is in good agreement with previous reports affirming the conservation of the CME network across Animalia (Schmid & McMahon, 2007) and might be viewed as a hallmark of the central role of this pathway in the correct performance of the cell.
Adaptor protein complexes and accessory proteins. Endocytic adaptors bridge interactions between the lipid phosphatidylinositol (4,5)-biphosphate [PtdIns(4,5)P2] anchored in the plasma membrane, clathrin and specific signals located within the tails of transmembrane receptors. Depending on the number of polypeptides in the adaptor complexes, two classes have been defined: (1) multimeric adaptor proteins which contain four polypeptides (α, β, γ, δ, µ or σ); and (2) the monomeric clathrin-associated sorting proteins (CLASPs) which are often referred to as accessory proteins (Maldonado-Baez & Wendland, 2006). A complete set of the genes which comprise the different subunits of the three multimeric adaptor proteins (AP1-AP3) are present in the pea aphid genome (AP4 complex is lacking in both D. melanogaster and A. pisum genomes). While AP2 is the principal adaptor of the endocytic process occurring at the plasma membrane (Slepnev & De Camilli, 2000), the other adaptor complexes are involved in vesicular trafficking from the trans-golgi network, the endosomal compartment or in the basolateral pathway (Owen et al., 2004).
Accessory proteins are involved in multiple steps of this pathway including membrane binding or bending (Wendland, 2002). For example, AP180, has a clathrin-cage assembly activity and promotes the formation of uniformly sized clathrin-coated vesicles (Zhang et al., 1998; Traub, 2003). The gene that encodes this protein could not be located within the A. pisum and the D. melanogaster genomes. However, a homologous protein (PiCalm) shown to perform a similar function in D. melanogaster was successfully identified (Table S1). Other accessory proteins, like epidermal growth factor pathway substrate 15 (Eps15) and Intersectin act as scaffolding proteins and, thus, represent organizing proteins (Tebar et al., 1996). As in D. melanogaster, only one member of each family is present in A. pisum genome (Table S1). A number of accessory molecules functioning as alternative adaptors for specific transmembrane receptors (Wendland, 2002) were conserved in A. pisum as compared with D. melanogaster: this concerns Numb and Arrestin genes encoding alternative adaptors for Notch and activated G-protein-coupled receptors (Table S1). On the contrary, disparities between D. melanogaster and A. pisum were found with the low-density lipoprotein receptor adaptor protein 1 (Ldlrap1, also known as ARH) and the ced-6 (homolog of human GULP1) genes which act as alternative adaptors for the low-density lipoprotein receptor. To ensure proper annotation, sequence homology was confirmed by multiple sequence alignment with similar sequences derived from other insects (Fig. 2). Ldlrap1 homolog was identified in A. pisum but not in D. melanogaster. In contrast, a homologous D. melanogaster ced-6 gene was not detected in the pea aphid genome. The presence of the Ldlrap1 gene and absence of the ced-6 gene has also been documented in one other insect genome (Tribolium castaneum), but not in the other insect genomes that have been described thus far (Table S2). These genes encode proteins containing the phosphotyrosine-binding domain (PTB) that attaches to the cytoplasmic tails of the LDL receptor and PtdIns(4,5)P2 (Eden et al., 2007). The redundancy of the PTB-binding domain-containing proteins may explain the partial conservation of these proteins in different insect species. Ced-6 proteins have also been shown to be essential for engulfment of apoptotic cells (Liu & Hengartner, 1998). These proteins may participate in lipid transport along with two other proteins found within the A. pisum genome, Dab2 and Numb.
Figure 2. Phylogenic analyses of Phosphotyrosine-binding-domain proteins. The phylogenetic tree was generated as described in the Experimental procedure section. The numbers on the branches represent the percentage of 1000 bootstrap pseudosamples supporting that branch; only values >40% are shown. Sequences from Acyrthosiphon pisum, Aedes aegypti, Anopheles gambiae, Apis mellifera, Bombix mori, Culex pipiens quinquefasciatus, Drosophila melanogaster, Homo sapiens, Nasonia vitripennis and Tribolium castaneum were used.
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Finally, amphysins and dynamins aid in the separation of the vesicle from the plasma membrane and Hsc70, Auxilin and endophilins promote uncoating (Brodsky et al., 2001; Conner & Schmid, 2003). With the exception of dynamins (see below), these genes display a high degree of conservation with those found in D. melanogaster (Table S1). Annotation of Hsc70 is described elsewhere (Gerardo et al., 2009).
Dynamins: a novel type only present in aphids. Dynamins are large GTPases involved in various processes including endocytosis and budding of transport vesicles, which are crucial for the circulative mode of virus transmission (Cherry & Perrimon, 2004; Praefcke & McMahon, 2004). Their basic function appears to be to constrain the shape of the lipid membrane to perform fission or fusion. Animals including insects typically have three types of dynamins (Dyn, Drp1, and Opa1) (Miyagishima et al., 2008). In many cases, a single gene corresponds to each type.
Screening of the A. pisum genome revealed 15 putative dynamin genes (Nakabachi & Miyagishima, 2010) (Table 1). In addition to a single orthologue for each of Dyn, Drp1, and Opa1 that are common in metazoa, 12 genes encoding a novel type of dynamins, not yet identified in any other organism, were found. Expressed sequence tag (EST) analyses and reverse transcription (RT)-PCRs showed that at least 11 of these novel type genes as well as three canonical genes are transcribed, suggesting that they are functional. Real-time quantitative RT-PCR further demonstrated that expressions of four out of 12 novel type dynamin genes are highly upregulated in the midgut, through which aphids take in phloem sap diets and plant viruses (Nakabachi & Miyagishima, 2010). As this type of dynamin is absent from all other fully sequenced organisms, the products of these genes may function in processes that are unique to aphids.
Actins. Actin is one of most highly conserved and abundant proteins in the cell. It is a ubiquitous protein throughout the eukaryotic system with high sequence similarity among species (Kaksonen et al., 2006). Actin was first described in muscle cells (Halliburton, 1887) but today actin is known to participate in a large array of functions. More importantly, the actin cytoskeleton interacts with various endocytic components (Apodaca, 2001), resulting in inter- or intra-cellular transport or relocation of the endogenous macromolecules as well as viruses (Ploubidou & Way, 2001). For example, actin protein interacts with dynamin at the neck region of the budding vesicle after plasma membrane invagination (Merrifield et al., 2002). Moreover, M. persicae actin was shown to interact with the luteovirid Beet western yellow virus (BWYV) (Seddas et al., 2004).
Actin also has an essential role in exocytosis. The actin filament creates a physical barrier which prevents trafficking or docking of the secretory materials towards the plasma membrane. Therefore, actin filaments have to be removed or relaxed from the plasma membrane to allow exocytosis (Miyake et al., 2001). Disruption of the actin cytoskeleton results in increased exocytosis (Jog et al., 2007), but complete actin depolymerization, inhibits exocytosis (Muallem et al., 1995).
Six actin genes have been identified in D. melanogaster (Tobin et al., 1980): Act88F and Act79B are adult muscle-specific, Act57B and Act87E are larval muscle-specific and Act5C and Act42A are cytoplasmic. Additionally, nine actin-related (ARP) genes are also described in the D. melanogaster genome. Four potential actin genes and 13 actin-related genes were identified in the A. pisum genome (Table 1). No orthologous genes for the muscle-specific Act79B, Act88F, Act57B or Act87E were found within the A. pisum genome, but instead, two different genes were identified, Act2 and Act3 (Fig. S1). The difference observed between A. pisum and D. melanogaster may be related to insect development (hemimetabolism for A. pisum and holometabolism for D. melanogaster). Two genes, Act1 and Act4, are similar to the cytoplasmic actin genes of D. melanogaster, Act5C and Act42A (Fig. S1). The cytoplasmic actin is believed to be implied in different functions and in particular in virus transcytosis. In spite of its name, the D. melanogaster gene Arp53D is a conventional actin protein (Muller et al., 2005). Arp53D orthologues are only found in other Drosophila species. The other 13 A. pisum genes encode for ARPs.
ARPs were discovered in the 1990s in eukaryotic cells. Presently, 11 ARP subfamilies (ARP1 to ARP11) have been defined according to their similarity to conventional actin sequences, with ARP1 being the most similar and ARP 10 the least similar. ARP11 was discovered after this classification was established and has higher similarity with conventional actin than ARP8. ARP1-ARP3, ARP10 and ARP11 are localized in the cytoplasm where they participate in actin assembly and movement of vesicles along microtubules (Schafer & Schroer, 1999). The other ARPs are predominantly localized in the nucleus where they participate in chromatin remodelling, DNA repair and regulation of transcription (Blessing et al., 2004). Other orphan ARPs have been identified in some organisms (Muller et al., 2005). Alignment of ARPs with conventional actins identified, for each ARP subfamily, the location of hotspots for insertions and deletions; for 8 ARP subfamilies (ARP1, ARP2, ARP3 and ARP5 to ARP9) discriminating motifs and single residues were found (Muller et al., 2005). Potential A. pisum ARP genes were assigned to different ARP subfamilies using the identified discriminating motifs. As in D. melanogaster ARP1 to ARP6, ARP8 and ARP11 genes were identified, no ARP7, ARP9 or ARP10 were found (Fig. S1). The results obtained for both insects are in accordance with the phylogenetic distribution of ARP genes. The absence of ARP7, ARP9 and ARP10 is not surprising because these genes are restricted to fungi (Muller et al., 2005). Four potential ARP4 genes were found in A. pisum, and partial EST coverage was identified for only one of them. This gene is also duplicated in Apis mellifera and T. castaneum. Two additional ARP genes were found within the A. pisum genome, ARPA and ARPB (Fig. S1). These genes appear to be unique to A. pisum, no homologues were identified in any other organism. These genes could not be assigned to any subfamily using the discriminating motifs, they are probably orphan ARPs.
Seventeen key reference residues involved in nucleotide binding have been identified (Muller et al., 2005). Conventional actins and ARP1–ARP3 (cytoplasmic ARPs) have more than 60% identical residues and 90% similar residues, and are able to bind ATP, whereas the rest of the ARPs (ARP4–ARP11), with fewer identical and similar residues might not bind ATP or bind with less affinity or through other residues. The two orphan ARPs have a low percentage of identical and conserved amino acids for the 17 key residues; they might be unable to bind ATP.
No transcriptomic data are available verifying the expression of these genes in A. pisum or in other aphids. Therefore, more experiments are needed to verify whether these genes are functional or not, and whether they are ‘aphid-exclusive’ or are more extensively widespread.