Auxilin (short for Putative Tyrosine-Protein Phosphatase Auxilin) plays a prominent role in the uncoating of CCVs in neurons. The gene encoding Auxilin, DNAJC6, is located on the short arm of chromosome 1 (1p31.3) and has multiple different isoforms produced by alternative splicing (Figure 1.12). Two protein-coding isoforms (ENST00000371069, ENST00000395325) account for the majority of DNAJC6 expression. Each is made up of 19 exons, with an alternative exon 1, and result in an Auxilin protein of 970 and 913 amino acids, respectively (Figure 1.12) (Lonsdale et al., 2013). It is unclear whether these two isoforms exert physiological differences in function, but the known functional domains of Auxilin are unaffected by the alternative splicing of the first exon. DNAJC6 expression is restricted to brain (Lonsdale et al., 2013) and is most abundantly expressed in neurons (Zhang et al., 2016a) (Figure 1.12).
At the molecular level, Auxilin contains an N-terminal Phosphatase and Tensin Homologue-like (PTEN-like) domain for lipid binding, followed by an unstructured hinge region, a clathrin binding domain and a C-terminal J-domain for HSC70 binding (Figure 1.14).
ENST00000395325.3 ENST00000371069.4 ENST00000494710.2 ENST00000483402.1 ENST00000463018.1 ENST00000263441.7 ENST00000498720.1 ENST00000472787.1 Brain - F ron tal Cortex (BA9) Brain - Cereb ellum Brain - Cereb ellar Hemisphe re Brain - S
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Figure 1.12: Tissue expression profile of DNAJC6 isoforms A Tissue expression profile of DNAJC6 isoforms. Dotted box indicates expression in brain. B Alternative splicing and exons making up the different DNAJC6 isoforms. Adapted from GTEx Analysis release V7 (Lonsdale et al., 2013).
Uncoating of CCVs very rapidly succeeds the pinching off of CCVs from the plasma membrane (Lee et al., 2006; Massol et al., 2006; Taylor et al., 2011). Biochemical assays established that this uncoating reaction is dependent on the presence of both ATP and the ATPase HSC70 (Braell et al., 1984; Schlossman et al., 1984; Ungewickell, 1985). Auxilin was found to be required as a co-chaperone for the HSC70-mediated clathrin uncoating of CCVs in the brain (Ahle and Ungewickell, 1990). The onset of the uncoating reaction is marked by a burst of Auxilin to the CCVs after pinching off the membrane (Lee et al., 2006; Massol et al., 2006). The timing of these events is important, as early recruitment could lead to the disassembly of an unfinished clathrin coat.
The PTEN domain of Auxilin mediates binding to phosphatidylinositols, and is required for the timely recruitment of Auxilin to CCVs (Figure 1.14) (Guan et al., 2010; Lee et al., 2006; Massol et al., 2006). The plasma membrane is chiefly composed of PI(4,5)P2, and smaller amounts of PI4P. A recent study by
the Kirchhausen lab has demonstrated that the budding of a CCV during CME results in a rapid burst of PI4P, PI3P (phosphatidylinositol 3 phosphate) and PI(3,4)P2 (phosphatidylinositol 3,4 biphosphate) on the CCV (He et al., 2017). The fission of a CCV results in membrane discontinuity that inhibits free exchange and lateral diffusion of phosphatidylinositols along the membrane. The activity of multiple kinases and phosphatases present on CCVs, including Synaptojanin 1, can thus result in a rapid conversion of phosphatidylinositols on the membrane of CCVs (He et al., 2017). Indeed, a burst of Synaptojanin 1 has been shown to precede the burst of Auxilin recruitment and the onset of CCV uncoating (Lee et al., 2006). Remarkably, Auxilin has been shown to have a binding preference for PI4P, PI3P and PI(3,4)P2 over other phosphatidylinositol species (Massol et al., 2006). Thus, the PTEN domain of Auxilin essentially serves as a coincidence detector of phosphatidylinositols, that senses the release of CCVs from the plasma membrane by its lipid content.
In addition to the PTEN domain, Auxilin also requires binding to clathrin for its correct localization to CCVs (Figure 1.14) (Fotin et al., 2004b), as neither domain by itself is sufficient for timely recruitment to newly budded CCVs (He et al., 2017; Lee et al., 2006; Massol et al., 2006). Auxilin contains multiple clathrin binding motifs that allow interaction with the terminal domain and distal leg of clathrin triskelia (Scheele et al., 2001; Scheele et al., 2003). The assembly of the clathrin coat is of such nature that three β-propeller domains are placed underneath the tripod of a clathrin triskelion (Figure 1.5), and each propeller domain interacts with a single Auxilin molecule (Fotin et al., 2004b; Smith et al., 2004; Xing et al., 2010).
The precise placement of Auxilin molecules results in the recruitment of HSC70-ATP, through interaction with the J-domain of Auxilin, to the proximity
of the triskelion tripod (Fotin et al., 2004b). The binding of Auxilin induces in a slight distortion of the clathrin coat, which allows HSC0-ATP to bind with hydrophobic motifs on the carboxy-termini of the clathrin heavy chains that protrude inward from the tripod (Rapoport et al., 2008). The ATPase activity of HSC70 is stimulated by this peptide binding and Auxilin binding (Holstein et al., 1996; Ungewickell et al., 1997), and the resulting conversion of HSC70-ATP to HSC70-ADP tightens the HSC70 clamp with the hydrophobic clathrin motif, locking the local distortion of the clathrin coat into place (B¨ocking et al., 2011; Fotin et al., 2004b; Xing et al., 2010). It is thought that a local distortion of a critical number of clathrin triskelia results in sufficient strain to destabilize the entire clathrin lattice for the subsequent uncoating of the CCV (B¨ocking et al., 2011; Xing et al., 2010).
Auxilin has also been found to interact with AP2 through a low affinity interaction of ‘DPF’ motifs within the clathrin binding domain (Kametaka et al., 2007; Owen et al., 1999; Scheele et al., 2001; Scheele et al., 2003). This interaction is not required for its recruitment to AP2 positive CCVs, but it is plausible that it helps stabilizing its interaction in a CCV and contributes to distinguish free and polymerized clathrin triskelia (Scheele et al., 2003).
Altogether, the role for Auxilin in the uncoating of CCVs has been well established. However, the Auxilin-dependent dynamics of uncoating in vivo have nearly exclusively been studied in the context of CME. An important open question is therefore to what extent its function can be extrapolated to clathrin-mediated trafficking at the Golgi apparatus. CCVs derived from the TGN differ from those derived from the plasma membrane, as they contain different adaptor proteins and are derived from membranes with different lipid compositions. Remarkably, Auxilin has been found to interact with plasma membrane-resident adaptor protein
AP2, but not with Golgi-resident AP1 or the GGAs (Kametaka et al., 2007). In addition, it is unclear whether the PTEN domain of Auxilin similarly senses the release of CCVs from the TGN by their lipid content as has been described for CME. Whereas the plasma membrane is chiefly composed of PI(4,5)P2, PI4P appears to be the principal phosphatidylinositol on the TGN (Daboussi et al., 2012b). It remains to be determined whether CCV budding from the TGN is similarly accompanied by a rapid conversion of phosphatidylinositol contents as is the case for CME.