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Gene names - stoned A and stoned B Synonyms - Cytological map position - 20A4--5 Function - synaptic vesicle endocytosis Keywords - neuromuscular junction, synapse, synaptic vesicle recycling |
Symbol - stnA and stnB FlyBase ID: FBgn0016976 and FBgn0016975 Genetic map position - Classification - novel protein (StnA) and clathrin adaptor protein-like (StnB) Cellular location - cytoplasmic |
After a nerve fires, proteins encoded by the stoned locus help ready the synapse for the next round of firing. stoned was identified several decades ago on the basis of stress-sensitive behavioral mutants (Grigliatti, 1973). The Drosophila stoned locus is dicistronic, that is, the locus encodes two distinctive presynaptic proteins, Stoned A (StnA) and Stoned B (StnB) from a single mRNA polymer. StnA is a novel protein without homology to known synaptic proteins, and StnB contains a domain with homology to the endocytotic protein AP50. After synaptic vesicle exocytosis during neural firing, the coordinated endocytosis of synaptic vesicle membrane proteins is mediated by adaptor proteins. Both Stoned proteins colocalize precisely with endocytotic proteins including the AP2 complex and Dynamin in the 'lattice network' characteristic of endocytotic domains in Drosophila presynaptic terminals. Dye uptake studies in stoned mutants demonstrate a striking decrease in the size of the cycling synaptic vesicle pool and loss of spatial regulation of the vesicular recycling intermediates. Mutant synapses display a significant delay in vesicular membrane retrieval after depolarization and neurotransmitter release. These studies suggest that the Stoned proteins play a role in mediating synaptic vesicle endocytosis (Fergestad, 2001; Stimson, 2001; Phillips, 2000).
A highly specific synaptic mislocalization and degradation of Synaptotagmin occurs in stoned mutants. Overexpression of Synaptotagmin rescues stoned embryonic lethality and restores endocytotic recycling to normal levels. Overexpression of Synaptotagmin I in otherwise wild-type animals results in increased synaptic dye uptake, indicating that Synaptotagmin directly regulates the cycling synaptic vesicle pool size. In vitro interaction studies indicate a physical interaction between Stoned B and Synaptotagmin. The Stoned A protein is also found in association with vesicles, and it too exhibits an in vitro association with Synaptotagmin. However, the bulk of Stoned A is in a nonmembranous fraction. These studies suggest that Stoned proteins regulate the AP2-Synaptotagmin interaction during synaptic vesicle endocytosis. It has been concluded that Stoned proteins control synaptic transmission strength by mediating the retrieval of Synaptotagmin from the plasma membrane (Fergestad, 2001; Stimson, 2001, and Phillips, 2000).
Since the evidence for physical interaction between Stoned proteins and Synaptotagmin has now gained acceptence, it is interesting to look back at the genetic evidence for an involvement of Stoned proteins in the recycling of Synaptogamin. This genetic evidence consists of an examination of the effects of stoned mutation on the localization of Synaptogamin, carried out by Fergestad (1999), and described in detail below. These results suggest that the Stoned proteins are essential for the recycling of synaptic vesicle membrane and are required for the proper sorting of Synaptotagmin during endocytosis.
Several lethal stoned alleles have been identified, including two transposable element insertions, stn13-120 and stnPH1, that lie in the StnA and StnB reading frames, respectively, and an EMS-induced allele, stnR9-10, that has not been characterized at the molecular level. All of these lethal stoned mutants die as mature embryos after a failure to hatch from the egg case, apparently from lack of coordinated movement. This defect is not a result of alterations in gross embryonic morphology; mutant embryos show normal segmental patterning of the epidermis, muscles, and nervous system. Thus, the mutant embryos appear morphologically normal but are impaired in the ability to move in a coordinated manner (Fergestad, 1999 and references therein).
Wild-type and mutant embryos (22-24 hr) were labeled with each of the Stoned antibodies to determine the effect of each mutation on protein expression and localization. At the embryonic wild-type NMJ, StnA and StnB proteins are highly concentrated in presynaptic boutons. None of the stoned mutant alleles display detectable StnB staining in the synaptic terminal, including the viable stnC embryo and the stnC third instar larva. In contrast, the mutants show variable levels of StnA expression. Both the stn13-120 and stnR9-10 alleles have severely reduced or undetectable levels of StnA expression, whereas the stnPH1 allele appears to have only moderately reduced levels of StnA. The viable stnC mutant NMJ also shows strongly reduced or undetectable StnA staining at 22 hr AF; however these animals do display very weak StnA expression at the third instar NMJ. These results suggest that a mutation in the first open reading frame of the dicistronic stoned locus (StnA) renders the second reading frame (StnB) unreadable. Thus, the stnPH1 allele primarily removes the StnB product, consistent with the transposable element insertion in the second reading frame (StnB), whereas all other alleles strongly effect the expression of both StnA and StnB (Fergestad, 1999).
Using a number of immunological markers, stoned mutant NMJs appear morphologically and molecularly similar to those of wild-type, although the terminals appear slightly smaller than normal. Despite this slight structural difference, the mutant NMJs display clear, punctate expression of several vesicle-associated proteins in the synaptic boutons, including CSP and Syb. In addition, the expression of other synaptic markers, including a neuronal membrane marker (HRP), syntaxin (Syx), and Rab3, appear normal in the mutant terminals. The quantified expression level and bouton localization of the synaptic proteins are similar to those of wild type (Fergestad, 1999).
In contrast, Synaptotagmin (Syt) protein appears to be strikingly mislocalized in all four stoned mutant alleles. Instead of the punctate bouton localization of Syt observed in wild type, the stoned mutants display reduced Syt expression in the boutons and the protein aberrantly dispersed throughout the presynaptic terminal. This mislocalization is not resolved with development in the viable stnC allele because Syt expression remains mislocalized at the third instar NMJ. Double-labeling assays with other presynaptic markers indicate this mislocalization is specific to Syt, because CSP, the neuronal membrane marker HRP, another synaptic vesicle protein (Syb), and the membrane protein Syx display normal patterns of expression in all mutants. These results suggest that stoned mutants specifically mislocalize the SV (synaptic vesicle) protein Syt in the synaptic terminal and retain other synaptic proteins properly (Fergestad, 1999).
For all four mutant alleles, the intensity of the Syt and CSP expression in individual, double-labeled boutons is quantified using digital confocal imaging and compared with wild-type expression levels in parallel trials. All mutants show an equal and similar ~40%-60% loss of Syt synaptic localization, compared with the normal expression and localization of CSP. This reduction in Syt expression is significant in all four alleles, and the alleles are not significantly different from each other. Syt expression in whole embryos was analyzed to determine further the nature of the synaptic loss of Syt staining. Quantified Western blots were performed to determine Syt expression levels in the different stoned alleles. Mutants display a significant decrease in Syt levels, with protein levels in the range of 20%-60% of those found in wild-type embryos. The decrease in in situ synaptic localization is consistent with the loss of Syt displayed on Western blots. These findings suggest that Syt is not only mislocalized in stoned mutants but may also be subject to rapid degradation when not properly localized (Fergestad, 1999).
To determine whether the striking synaptic staining pattern for the stoned proteins is consistent with a physiological function at the synapse, transmission properties were assayed with electrophysiological recordings at the embryonic NMJ. In all stoned mutant alleles, nerve stimulation produces muscle contraction, demonstrating that presynaptic depolarization evokes transmitter release and that the muscle excitation-secretion response is intact. However, evoked excitatory junctional currents (EJC) peak amplitudes are significantly reduced below wild-type levels, typically by 30%-50%, for all stoned alleles. Furthermore, the release of neurotransmitter at mutant synapses is markedly asynchronous. The asynchronous mutant transmission seems to result from delayed presynaptic vesicle fusion, similar to that observed for the previously identified synaptotagmin mutant (Fergestad, 1999).
The reduced and erratic evoked transmission of stoned mutants is further increased after prolonged, repetitive stimulation at moderate or high frequencies (5-20 Hz). To determine the nature of this debilitation, animals were subjected to a high-frequency stimulus protocol (10 Hz) sustained over a 5 min period. The average EJC amplitude at wild-type NMJs decreases by an absolute amount similar to that of mutant animals (~500 pA), suggesting that the NMJ of both genotypes is fatiguing comparably. However, the stoned mutants start with significantly impaired performance and fatigue by an average of 50-75% during the stimulus train, whereas wild-type amplitude decreases by only ~20%-25%. The decrease in mean EJC amplitude is accompanied by a large increase in transmission failure in the stoned synapses. Wild-type synapses maintain high-amplitude, high-fidelity transmission over a sustained stimulation period of 5 min and show no failures even at the end of the stimulus train. In contrast, the failure frequency of the mutant synapses is initially significant (~5%-10%) and increases rapidly during sustained stimulation to a level of 25%-80% at the end of the stimulus train. The three alleles that more strongly affect both StnA and StnB expression (stnR9-10, stn13-120, and stnC) show a more marked transmission failure rate (50%-80%) than does the primarily StnB mutant (stnPH1; 25%). The striking increase in failure rate during a prolonged stimuli train suggests that repetitive stimulation results in a severe depletion of SVs in the stoned mutants (Fergestad, 1999).
The Drosophila embryonic NMJ is characterized by the presence of presynaptic varicosities (boutons) containing specialized, densely staining, T-shaped structures (t-bars) at the presynaptic active zones, the putative SV fusion sites. The pre- and post-synaptic membranes surrounding the t-bars are densely stained and are separated by a cleft ~15 nm wide. Clear SVs of 30-40 nm diameter are observed clustered around the t-bars in a semicircular area with a radius of ~250 nm. Synaptic vesicles dock with the membrane immediately adjacent to t-bars in preparation for evoked fusion. For analytical purposes, vesicles are considered docked if distributed less than one vesicle diameter (<30 nm) from the plasma membrane at active sites. Synaptic vesicles also appear outside the clusters surrounding t-bars, although at a much lower density than that of the clustered vesicles. Other membrane structures, such as large dense core vesicles and translucent vesicles larger than typical SVs (presumed to be endosomes), are also occasionally observed in sections through boutons containing t-bars (Fergestad, 1999 and references therein).
Striking differences in synaptic ultrastructure are observed for all of the stoned mutant alleles relative to wild-type controls. Presynaptic boutons containing t-bars are present in all stoned alleles, and the association of presynaptic tissue with muscle cells is similar to that in wild type, indicating that development of NMJs in the mutant embryos occurs normally. However, a striking reduction in the number and density of SVs present in boutons is observed in stoned mutants. All four stoned alleles have ~50% fewer SVs clustered around the active zone t-bars, and SV density outside of the clustered radius surrounding t-bars is also reduced by ~50%. Analysis of stnPH1/Df(1)HM430 animals showed that they displayed features identical to those of animals hemizygous for stnPH1. A similar 50% reduction in the number of docked vesicles per t-bar is observed at stn13-120, stnR9-10, and stnPH1 synapses, whereas the viable stnC allele has ~30% fewer docked vesicles per t-bar than does wild type. Thus, SV density is severely reduced in all mutant alleles, throughout the presynaptic bouton, at active zones, and in the number of docked vesicles (Fergestad, 1999).
An additional difference observed in stoned bouton ultrastructure is an increase in intermediates of the SV cycle. In wild-type embryos, SVs are the most prominent membrane structures in sections through boutons containing t-bars. A much smaller number of translucent vesicles (cisternae) noticeably larger than SVs and early endosomes are also present in these sections, and, rarely, multivesicular bodies (MVBs) are seen. The early endosomes represent a step in the normal synthetic pathway for SVs in these terminals. Increased numbers of MVBs result from increased synaptic activity. These MVBs are usually removed from the region of the active zone and are targeted to somatic lysosomes. Because MVBs have been shown to contain SV proteins, they represent the normal degradative route for synaptic proteins (Fergestad, 1999).
In stoned mutants, sections through boutons containing t-bars have a significantly greater number of large vesicles (>60 nm; endosomes and cisternae) than do wild-type synapses. In addition, a greater number of these large vesicles appear within the SV cluster surrounding active zones in mutant embryos. The number of large vesicles present within the clustered radius of t-bars is at least three times greater in all of the mutant strains than in wild type. Furthermore, a significant increase in the number of MVBs is observed in sections through boutons containing t-bars in stoned mutants. stnPH1 and stnC have an approximate fourfold increase in the number of MVBs compared with that in wild type (Fergestad, 1999).
These results indicate that the stoned mutants have normal gross synaptic morphology; however, these mutants display a severe reduction in synaptic vesicle number and an increase in recycling intermediates including large cisternae and MVBs. This ultrastructural analysis combined with the abnormal labeling of synaptotagmin and debilitated synaptic transmission strongly suggests a role for the stoned proteins in regulating the synaptic vesicle-recycling pathways.
Early models of synaptic membrane recycling suggested that newly endocytosed vesicles join a sorting endosome compartment before subsequent budding and maturation. In addition, under periods of sustained or high-frequency transmission, large patches of membrane may be retrieved from the plasma membrane to form an early endosome from which new vesicles may be generated. Recent studies provide evidence that synapses may also recycle synaptic vesicle membrane directly, without first fusing with an endosomal-sorting compartment. Evidence from Drosophila argues that these two recycling pathways may act in parallel, corresponding to functionally and spatially distinct vesicle pools. The stoned mutants are clearly defective in one or more of these recycling pathways. All alleles show a significant decrease in SVs at active zones and throughout the presynaptic terminal and a correspondingly increased sequestering of membrane in enlarged membranous compartments. These defects may result from a direct defect in endocytosis leading to improper regulation of vesicle size, or alternatively, the large vesicles may be endosomal compartments that accumulate owing to an inability to bud and segregate new SVs during endosome-mediated recycling. The existence of a population of very large-amplitude spontaneous fusion events (200-500 pA) at stoned synapses suggests that these large vesicles can function to release neurotransmitter in a constitutive manner (similar to large dense-core vesicles). However, the apparent functional competence of these vesicles does not allow for a determination of whether they represent abnormally large SVs or endosomes that have spilled over into the active zone. The accumulation of MVBs may be more informative. These structures clearly derive from endosomes and represent a normal degradative pathway in which SV proteins and membrane are targeted to somatic lysosomes. In parallel with MVB accumulation, Syt levels at the terminal and throughout the embryo decrease by ~50% in stoned mutants. It is therefore suggested that the Stoned proteins are specifically involved in the recruitment and localization of Syt during SV recycling and, in the absence of correct targeting, that Syt is degraded via a default degradative pathway involving MVBs (Fergestad, 1999).
Mature synaptic vesicles have a specific complement of proteins required for a variety of functions. Each protein must be selectively recruited to the maturing SV during endocytosis. The mislocalization of Syt in stoned synapses is not accompanied by a loss of other SV proteins (e.g., synaptobrevin and CSP), suggesting that the stoned proteins may be involved in the specific recycling of Syt from endocytosed membrane. It is hypothesized that the Stoned proteins normally function at a choice point segregating recycled Syt protein into maturing synaptic SVs and away from the MVB degradative pathway. Such a role has been suggested for the AP3 complex, shown recently to be required for localization of a SV transporter protein and to be required for the generation of SVs. Similarly, the Drosophila gene LAP, which encodes AP180 (associated with clathrin-dependent endocytosis with the AP2 complex), has been shown recently to be involved in regulating SV size and the proper recruitment of the vesicle coat protein clathrin (Zhang, 1998). In the C. elegans AP180 mutant (UNC-11), the protein also seems to have a specific role in recruiting synaptobrevin to the recycled vesicle (E. Jorgensen, personal communication to Fergestad, 1999). These AP180 data, combined with the observation that SV size is not altered in mutant animals lacking synaptobrevin, suggest that AP180 has two distinct functions: structural budding of membrane and the specific recycling of synaptobrevin (Fergestad, 1999).
The similarities between these studies led to the hypothesis that there may be separate mechanisms required for the recycling of each distinct SV protein and that these mechanisms may be intimately integrated into the membrane-budding machinery. Such a coupled mechanism would guarantee that newly generated SVs have the correct functional complement of SV proteins. Clearly, within this general mechanism, the Stoned proteins couple the specific recruitment of Syt to proper SV biogenesis. The Stoned proteins may participate in the AP2-mediated plasma membrane mechanism or, alternatively, act in a separate and/or later site such as AP3-mediated endosomal sorting to direct the recruitment and/or localization of Syt into mature SVs (Fergestad, 1999).
Retrieval of SV components from the plasma membrane is tightly temporally coupled to exocytosis. Mutation of stoned disrupts this temporal coupling and delays the onset and rate of endocytosis after exocytosis. Delayed application of FM1-43 to stoned synapses after stimulation reveals that the delayed component of endocytosis is comparable with wild-type levels. There are several possible explanations for these altered endocytosis kinetics in stoned mutants. A likely rationale is that global membrane retrieval is delayed in stoned mutants, suggesting that Stoned proteins may play a role in either initiating endocytosis or facilitating the speed of endocytosis. An alternative scenario may be that there are multiple pathways for SV endocytosis, which differ in temporal kinetics, and that the rapid endocytosis mechanism is specifically impaired in stoned mutants. Although it is formally possible that delays in exocytosis might also explain the delayed membrane retrieval, recordings of synaptic transmission show that exocytosis speed is only very minimally impaired in stoned mutants (Fergestad, 2001).
How are the numerous different components of the SV recognized and recombined in the precise stoichiometric ratios required for synaptic function? There is an increasing body of evidence that a cast of specific recycling proteins is required to recognize and retrieve specific components of the SV during plasma membrane endocytosis. At center stage, the AP2 complex plays a prominent role in clathrin-mediated SV endocytosis. Genetic removal of the alpha-Adaptin subunit in Drosophila shows that the AP2 complex is absolutely required for the SV endocytotic process in this system (Gonzalez-Gaitan, 1997). Moreover, the AP2 complex has been shown to bind Synaptotagmin (Zhang, 1994), and it thus seems likely that this complex mediates the endocytotic recovery of Synaptotagmin, likely in addition to other integral SV proteins (Fergestad, 2001).
It is proposed that the role of the Stoned proteins may be to recycle Synaptotagmin by mediating the association with the AP2 complex. It has been shown recently that both StnA and StnB bind with high specificity to Synaptotagmin (Phillips, 2000) and therefore likely act in a cooperative manner in Synaptotagmin retrieval. Studies reported by Haucke and De Camilli (1999) have shown that the AP2-Synaptotagmin interaction can be stimulated by the presence of Yxxphi-containing peptides, which also enhance the recruitment of AP2 to the plasma membrane. Because both Stoned proteins contain multiple copies of these tyrosine-based motifs, as well as other AP2 and Clathrin binding domains, it is hypothesized that the Stoned proteins specifically promote the retrieval of Synaptotagmin from the plasma membrane by mediating the AP2-Synaptotagmin binding. Because the Stoned proteins are encoded by a dicistronic locus, polarity constraints have to date prevented an independent dissection of the roles of StnA and StnB in Synaptotagmin endocytosis. Why does the process require two proteins, and what does each contribute to the recycling mechanism? Targeted homologous knock-out techniques are being used to explore these questions (Fergestad, 2001).
As the proposed calcium sensor for SV fusion, Synaptotagmin is likely to function as a key regulator of transmission strength. The number of Synaptotagmin proteins in an SV membrane may play an important role in regulating the response of the presynaptic terminal to depolarizing stimuli. Overexpression of Synaptotagmin alone is capable of substantially increasing the size of the endo-exo SV pool. Therefore, the Stoned proteins, by regulating Synaptotagmin recycling, also act as key regulators of neurotransmission strength. Future experiments are focusing on the specific regulation of Synaptotagmin levels by each of the Stoned proteins (Fergestad, 2001 and references therein).
The stoned gene of Drosophila is required for normal neuronal function in both adult and larva. DNA sequences have been identified that lie within a genetic region that is known to include the stoned gene and that also reveal restriction site variations in two stoned lethal mutants. This genomic region contains a single transcription unit coding for an approximately 8.4-kb transcript. The transcript is preferentially expressed in the head of adult flies. The isolation and sequencing of cDNA and genomic clones reveals that stoned appears to encode a dicistronic mRNA, although the possible existence of other forms of mRNA cannot be excluded. Antibody cross-reactivity shows that two proteins are translated from the stoned locus in vivo. Both open reading frames (ORFs) encode novel proteins. The protein encoded by the first ORF contains four tandemly repeated motifs, and one domain of the protein encoded by the second ORF shows similarity to a family of proteins (AP50s) associated with clathrin assembly protein complexes (Andrews, 1996).
StnA contains five DPF motifs (alpha-Adaptin-interacting motifs; a subunit of AP2). Furthermore, StnA also contains the Clathrin beta-propeller binding motif LL(D/E/N)phi(D/E) and four Yxxphi AP2 binding motifs. STNB has a region of partial homology with AP50, a subunit of the AP2 adaptor protein complex involved in SV endocytosis (De Camilli, 1996). Although this conserved domain has 42% amino acid identity with the AP50 family as a whole (Andrews, 1996), StnB is severalfold larger than AP50 and is only distantly related. Furthermore, StnB is not the Drosophila AP50 homolog because the Drosophila AP50 has recently been cloned with 86% homology to the human gene and is located at 94B1-2 (Zhang, 1999), suggesting that STNB does not act as part of the AP2 complex. StnB contains 12 Yxxphi AP2 binding motifs and a (D/E)xxxLL AP2 binding motif, all of which are thought to be involved in organizing the endocytotic machinery. StnB also has strong homology with unc-41, a gene identified in C. elegans on the basis of a neurological defect (uncoordinated) and believed to have a role in SV recycling (Cremona, 1997). StnB also contains a proline-rich N terminal (SH3 binding) region. In addition, the N terminal of the StnB protein contains four NPF motifs (Andrews, 1996). These sequences are bound by an Eps15 domain, found in proteins involved in endocytosis (Tebar, 1996; Salcini, 1997), and thus may regulate interactions of StnB with proteins such as Dap160 (Roos, 1998). The presence of the NPF motifs and the homology with UNC-41 and AP50 further suggest a role for the StnB protein in synaptic function and, in combination with the shibire genetic interaction, reinforce the idea of a possible role for the stoned proteins in synaptic vesicle endocytosis (Fergestad, 1999 and Fergestad, 2001 and references therein).
Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery, have been identified. The human stonins are homologous to the Drosophila melanogaster Stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery (Martina, 2001).
Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. The endocytotic protein stonin 2 has been shown to localize to axonal vesicle clusters through its micro-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to micro2. Mutation of the tyrosine motif-binding pocket of micro2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. It is hypothesized that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling (Walther, 2001).
Synaptic vesicle recycling is in part mediated by clathrin-mediated endocytosis. This process involves the coordinated assembly of clathrin and adaptor proteins and the concomitant selection of cargo proteins. The endocytotic protein stonin 2 localizes to axonal vesicle clusters through its mu-homology domain. Interaction of this domain with synaptotagmin I is sufficient to recruit stonin 2 to the plasmalemma. The N-terminal domain of stonin 2 harbors multiple AP-2-interaction motifs that bind to the clathrin adaptor complex AP-2. These motifs with the consensus sequence WVxF are capable of binding to the alpha-adaptin ear domain and to mu2. Mutation of the tyrosine motif-binding pocket of mu2 abolishes recognition of the WVxF peptide, suggesting that association with stonin 2 renders AP-2 incompetent to sort tyrosine motif-containing cargo proteins. It is hypothesized that stonin 2 may function as an AP-2-dependent sorting adaptor for synaptic vesicle recycling (Walther, 2004).
Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. This study shows that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. These data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling (Diril, 2006).
date revised: 26 May 2001
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