In this study, we have identified a functional role for the Troyer syndrome protein spartin in endocytosis. Although spartin is predominantly cytosolic, it is recruited to enlarged endosomes induced by Vps4DN. It is also redistributed from the cytoplasm to the plasma membrane immediately after EGFR activation and is then present in EGF-positive endosomes. After cells were depleted of spartin using specific siRNAs, they exhibited a decreased rate of EGFR degradation compared with control cells. In addition, depletion of spartin results in decreased uptake and/or recycling of EGF. Taken together, our data indicate that spartin is an important component in the intracellular sorting of EGFR and very likely other ubiquitinated receptors.
Low levels of EGF trigger the internalization of EGFR via clathrin-mediated endocytosis (Sigismund et al., 2005
). This complex, highly coordinated process begins with the recruitment of AP-2, an adaptor protein that drives the assembly of the clathrin lattice and binds to many accessory proteins, including amphiphysin, intersectin, epsins, auxilin, and Eps15 (Kirchhausen, 2000
; Slepnev and De Camilli, 2000
). Several lines of evidence support the role of Eps15 in endocytosis (Tebar et al., 1996
; Carbone et al., 1997
; Benmerah et al., 1998
; Benmerah et al., 1999
). Indeed, Eps15 is redistributed after EGFR activation; it is initially recruited to the plasma membrane and then follows the endocytosed EGFR (Torrisi et al., 1999
). In general, the behavior of spartin in response to EGFR activation is similar to that of Eps15. Presently, the molecular mechanisms for the redistribution of spartin are unknown. However, one possibility might be that, upon EGFR stimulation, phosphorylated Eps15 recruits spartin and translocates the spartin-Eps15 complex to the plasma membrane, most likely to coated pits, where the presence of Eps15 is well documented (Tebar et al., 1996
; Torrisi et al., 1999
). Alternatively, EGFR activation might induce phosphorylation of spartin, and this modification could be responsible for the translocation of spartin to the plasma membrane, where spartin might associate with its binding partner, Eps15. Although we have not detected phosphorylation of spartin on Tyr residues (data not shown), we are currently examining Ser/Thr phosphorylation of spartin.
An important finding of our study is that acute depletion of spartin by siRNA in HeLa cells decreases the rate of EGFR degradation. After internalization of EGFR, the occupied receptor is sorted to the late endosomes for subsequent degradation by the lysosomes, and depletion of many proteins known to be involved in sorting of EGFR results in the diminished degradation of this receptor. For example, down-regulation of Hrs or STAM1 and STAM2, which are mono-ubiquitinated and contain ubiquitin-interacting motifs, decreases EGFR degradation by 40 and 35%, respectively, compared with control cells (Bache et al., 2003
; Kanazawa et al., 2003
). The colocalization of spartin with EGFR for 30 min after its internalization, together with the data showing that depletion of spartin decreases the rate of EGFR degradation, indicates that spartin regulates the sorting of ubiquitinated cargo receptors.
Another major finding of this study is the identification of the covalent modification of spartin by ubiquitination. Using immunoblotting, we observed protein bands representing endogenous spartin at ~85 and ~95 kDa in different human cell lines, which concurs with another report using a different antibody (Robay et al., 2006
), and we demonstrated that the 95-kDa band represents a mono-ubiquitinated form of spartin. We estimate that ~30–35% of endogenous spartin is mono-ubiquitinated and that this modification does not depend on EGF stimulation, indicating that spartin constitutively undergoes this post-translational modification. Other investigators showed that mono-ubiquitination of endogenous or overexpressed Eps15 in Her14 cells (NIH3T3 cells expressing EGFR) occurs upon EGF stimulation (van Delft et al., 1997
; Klapisz et al., 2002
; Fallon et al., 2006
), whereas in other cell lines endogenous Eps15 is constitutively mono-ubiquitinated (Klapisz et al., 2002
). Mono-ubiquitination of other proteins involved in trafficking of internalized cargo is EGF dependent. For example, overexpressed Hrs, epsin1, and epsin2 in COS-7 cells have been shown to be ubiquitinated only in response to EGF stimulation (Polo et al., 2002
). It is reasonable to infer that although mono-ubiquitination of some sorting proteins is EGF dependent, other proteins are constitutively ubiquitinated. By this means they might present different molecular modules in the ubiquitin-receptor network and regulate specific aspects of clathrin-dependent or clathrin-independent endocytic pathways.
Similar to other mono-ubiquitinated proteins involved in endocytic trafficking of cargo receptors (Klapisz et al., 2002
; Oldham et al., 2002
; Polo et al., 2002
), spartin binds to the ubiquitin moiety. Moreover, like most proteins, spartin interacts with ubiquitin via its Ile44
hydrophobic pocket (Hicke et al., 2005
). Currently, at least 15 different ubiquitin-binding domains have been characterized, including ubiquitin-interacting motif, ubiquitin E2 variant domain, and ubiquitin-associated domain (Hicke et al., 2005
). However, spartin does not harbor any of these and thus likely contains a novel ubiquitin-binding region.
Knockdown of endogenous spartin or Eps15 in HeLa cells by siRNA, individually or in combination, decreases the rate of EGF uptake by ~30% compared with cells treated with control siRNA. Because we applied physiological, low levels of 125
I-EGF (1.5 ng/ml) which preferentially uses the clathrin-dependent pathway for EGFR internalization (Sigismund et al., 2005
), our data indicate that spartin, in conjunction with Eps15, may function in clathrin-mediated endocytosis of EGFR. These results also suggest that spartin might be involved in the internalization of EGFR. Alternatively, spartin depletion might instead influence the rate of EGFR recycling, which is known to have a half-time of ~5 min (Sorkin et al., 1991
). Future detailed biochemical assays as well as experiments examining the presence of spartin on coated-pits will clarify the role of spartin in the internalization and/or recycling of EGFR.
The SPG20 mutation in the spartin
gene results in a premature stop codon and possibly degradation of the truncated protein that results in loss of function and subsequently a length-dependent axonopathy of the corticospinal motor neurons, the cardinal feature of HSPs. This impairment might be caused by dysfunctions at pre- and/or postsynaptic structures, but at present we can only speculate about the function of spartin in neurons. A recent study found spartin in synaptic nerve terminals (Robay et al., 2006
), and the depletion of EHS-1, an ortholog of Eps15 in Caenorhabditis elegans
, resulted in a temperature-dependent defect in movement as well as a decreased number of synaptic vesicles (Salcini et al., 2001
). Thus, spartin, similar to Eps15, might be an accessory protein involved in clathrin-mediated endocytosis of synaptic vesicles in presynaptic nerve terminals. Another possible role for spartin in neurons may be in the internalization, sorting, and/or degradation of specific receptors for neurotrophic factors such as BDNF or NGF, which are important for cell survival and neurite outgrowth. Interestingly, a number of proteins mutated in neurological disorders, including alsin, huntingtin, and parkin, have roles in endocytosis (Devon et al., 2006
; Fallon et al., 2006
; Rong et al., 2006
). It remains to be determined whether lack of spartin affects endocytosis of growth factor receptors, which might lead to deficiency in survival signals and impaired neurite outgrowth.
In summary, we demonstrate the functional role of spartin in the regulation of clathrin-mediated endocytosis of EGFR. Future studies in neurons from spartin knockout mice, which we are currently generating, will assess the functional role of spartin in pre- and postsynaptic terminals and clarify mechanisms by which the lack of spartin results in a length-dependent axonopathy.