Evan Eisenberg

Multiple Roles of Auxilin and Hsc70 in Clathrin‐Mediated Endocytosis

The ATP‐dependent dissociation of clathrin from clathrin‐coated vesicles (CCVs) by the molecular chaperone Hsc70 requires J‐domain cofactor proteins, either auxilin or cyclin‐G‐associated kinase (GAK). Both the nerve‐specific auxilin and the ubiquitous GAK induce CCVs to bind to Hsc70. The removal of auxilin or GAK from various organisms and cells has provided definitive evidence that Hsc70 uncoats CCVs in vivo. In addition, evidence from various studies has suggested that Hsc70 and auxilin are involved in several other key processes that occur during clathrin‐mediated endocytosis. First, Hsc70 and auxilin are required for the clathrin exchange that occurs during coated‐pit invagination and constriction; this clathrin exchange may catalyze any rearrangement of the clathrin‐coated pit (CCP) structure that is required during invagination and constriction. Second, Hsc70 and auxilin may chaperone clathrin after it dissociates from CCPs so that it does not aggregate in the cytosol. Third, auxilin and Hsc70 may be involved in the rebinding of clathrin to the plasma membrane to form new CCPs and independently appear to chaperone adaptor proteins so that they can also rebind to membranes to nucleate the formation of new CCPs. Finally, if formation of the curved clathrin coat induces membrane curvature, then Hsc70 and auxilin provide the energy for this curvature by inducing ATP‐dependent clathrin exchange and rearrangement during endocytosis and ATP‐dependent dissociation of clathrin at the end of the cycle so that it is energetically primed to rebind to the plasma membrane.

Clathrin exchange during clathrin-mediated endocytosis

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein–clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.

Role of Cyclin G-associated Kinase in Uncoating Clathrin-coated Vesicles from Non-neuronal Cells

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.

A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo

Clathrin-coated vesicles mediate diverse processes such as nutrient uptake, downregulation of hormone receptors, formation of synaptic vesicles, virus entry, and transport of biosynthetic proteins to lysosomes. Cycles of coat assembly and disassembly are integral features of clathrin-mediated vesicular transport (Fig. 1a). Coat assembly involves recruitment of clathrin triskelia, adaptor complexes and other factors that influence coat assembly, cargo sequestration, membrane invagination and scission (Fig. 1a). Coat disassembly is thought to be essential for fusion of vesicles with target membranes and for recycling components of clathrin coats to the cytoplasm for further rounds of vesicle formation. In vitro, cytosolic heat-shock protein 70 (Hsp70) and the J-domain co-chaperone auxilin catalyse coat disassembly. However, a specific function of these factors in uncoating in vivo has not been demonstrated, leaving the physiological mechanism and significance of uncoating unclear. Here we report the identification and characterization of a Saccharomyces cerevisiae J-domain protein, Aux1. Inactivation of Aux1 results in accumulation of clathrin-coated vesicles, impaired cargo delivery, and an increased ratio of vesicle-associated to cytoplasmic clathrin. Our results demonstrate an in vivo uncoating function of a J domain co-chaperone and establish the physiological significance of uncoating in transport mediated by clathrin-coated vesicles.

Recruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis

Cyclin G-associated kinase (GAK), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for Hsc70-dependent uncoating of clathrin-coated vesicles. Total internal reflectance microscopy was used to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits. Following transient recruitment of dynamin to the clathrin puncta, large amounts of GAK are transiently recruited. GAK and clathrin then disappear from the evanescent field as the pit invaginates from the plasma membrane and finally these proteins disappear from the epifluorescence field, probably as the clathrin is uncoated from the budded vesicles by Hsc70. The recruitment of GAK is dependent on its PTEN-like domain, which we found binds to phospholipids. This suggests that interaction with phospholipids is essential for recruitment of GAK and, in turn, Hsc70, but Hsc70 recruitment alone might not be sufficient to induce irreversible clathrin uncoating. When budding of clathrin-coated pits is inhibited by actin depolymerization, there is repeated flashing of GAK on the clathrin-coated pit but neither scission nor irreversible uncoating occur. Therefore, budding as well as synchronous recruitment of GAK might be required for irreversible clathrin uncoating.

Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo.

The budding of clathrin-coated vesicles is essential for protein transport. After budding, clathrin must be uncoated before the vesicles can fuse with other membranous structures. In vitro, the molecular chaperone Hsc70 uncoats clathrin-coated vesicles in an ATP-dependent process that requires a specific J-domain protein such as auxilin. However, there is little evidence that either Hsc70 or auxilin is essential in vivo. Here we show that C. elegans has a single auxilin homologue that is identical to mammalian auxilin in its in vitro activity. When RNA-mediated interference (RNAi) is used to inhibit auxilin expression in C. elegans, oocytes show markedly reduced receptor-mediated endocytosis of yolk protein tagged with green fluorescent protein (GFP). In addition, most of these worms arrest during larval development, exhibit defective distribution of GFP–clathrin in many cell types, and show a marked change in clathrin dynamics, as determined by fluorescence recovery after photobleaching (FRAP). We conclude that auxilin is required for in vivo clathrin-mediated endocytosis and development in C. elegans.

The molecular chaperones Hsp90 and Hsc70 are both necessary and sufficient to activate hormone binding by glucocorticoid receptor.

Glucocorticoid receptors must be complexed with Hsp90 in order to bind steroids, and it has been reported that at least three other proteins, Hop, Hsc70, and a J-domain protein (either Hsp40 or Ydj1), are required for formation of active Hsp90-steroid receptor complex. In the present study, we reinvestigated activation of stripped steroid receptors isolated from either L cells or WCL2 cells. Surprisingly, we found, using highly purified proteins, that only Hsp90 and Hsc70 are required for the activation of glucocorticoid receptors in the presence of steroids; in the absence of steroids, either p23 or molybdate are also required as reported previously. Addition of Hop or Ydj1 had no affect on the rate or magnitude of the activation of the stripped receptors, and quantitative Western blots confirmed that neither Hop or Hsp40 were present in our protein preparations or in the stripped receptors. Furthermore, a truncated recombinant Hsp70 that does not bind Hop or Hsp40 was as effective as wild-type Hsp70 in activating stripped receptor. Since Hsc70 does not bind directly to Hsp90 but both proteins bind to Hop, it has been suggested that Hop acts as a bridge between Hsp90 and Hsp70. However, we found that after Hsc70 or Hsp90 bind directly to the stripped receptors, they are fully reactivated by Hsp90 or Hsc70, respectively. We, therefore, conclude that Hsp90 and Hsc70 bind independently to stripped glucocorticoid receptors and alone are sufficient to activate them to bind steroids.

Role for Hsp70 chaperone in Saccharomyces cerevisiae prion seed replication.

ABSTRACT The Saccharomyces cerevisiae [PSI+] prion is a misfolded form of Sup35p that propagates as self-replicating cytoplasmic aggregates. Replication is believed to occur through breakage of transmissible [PSI+] prion particles, or seeds, into more numerous pieces. In [PSI+] cells, large Sup35p aggregates are formed by coalescence of smaller sodium dodecyl sulfate-insoluble polymers. It is uncertain if polymers or higher-order aggregates or both act as prion seeds. A mutant Hsp70 chaperone, Ssa1-21p, reduces the number of transmissible [PSI+] seeds per cell by 10-fold but the overall amount of aggregated Sup35p by only two- to threefold. This discrepancy could be explained if, in SSA1-21 cells, [PSI+] seeds are larger or more of the aggregated Sup35p does not function as a seed. To visualize differences in aggregate size, we constructed a Sup35-green fluorescent protein (GFP) fusion (NGMC) that has normal Sup35p function and can propagate like [PSI+]. Unlike GFP fusions lacking Sup35ps essential C-terminal domain, NGMC did not form fluorescent foci in log-phase [PSI+] cells. However, using fluorescence recovery after photobleaching and size fractionation techniques, we find evidence that NGMC is aggregated in these cells. Furthermore, the aggregates were larger in SSA1-21 cells, but the size of NGMC polymers was unchanged. Possibly, NGMC aggregates are bigger in SSA1-21 cells because they contain more polymers. Our data suggest that Ssa1-21p interferes with disruption of large Sup35p aggregates, which lack or have limited capacity to function as seed, into polymers that function more efficiently as [PSI+] seeds.

ATP Binding Regulates Oligomerization and Endosome Association of RME-1 Family Proteins

Members of the RME-1/mRme-1/EHD1 protein family have recently been shown to function in the recycling of membrane proteins from recycling endosomes to the plasma membrane. RME-1 family proteins are normally found in close association with recycling endosomes and the vesicles and tubules emanating from these endosomes, consistent with the proposal that these proteins directly participate in endosomal transport. RME-1 family proteins contain a C-terminal EH (eps15 homology) domain thought to be involved in linking RME-1 to other endocytic proteins, a coiled-coil domain thought to be involved in homo-oligomerization and an N-terminal P-loop domain thought to mediate nucleotide binding. In the present study, we show that both Caenorhabditis elegans and mouse RME-1 proteins bind and hydrolyze ATP. No significant GTP binding or hydrolysis was detected. Mutation or deletion of the ATP-binding P-loop prevented RME-1 oligomerization and at the same time dissociated RME-1 from endosomes. In addition, ATP depletion caused RME-1 to lose its endosome association in the cell, resulting in cytosolic localization. Taken together, these results indicate that ATP binding is required for oligomerization of mRme-1/EHD1, which in turn is required for its association with endosomes.

Endocytosis and clathrin-uncoating defects at synapses of auxilin knockout mice

Neuronally expressed auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.