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.

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.

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.

HIV type 1 (HIV-1) was shown to assemble either at the plasma membrane or in the membrane of late endosomes. Now, we report an essential role for human ubiquitin ligase POSH (Plenty of SH3s; hPOSH), a trans-Golgi network-associated protein, in the targeting of HIV-1 to the plasma membrane. Small inhibitory RNA-mediated silencing of hPOSH ablates virus secretion and Gag plasma membrane localization. Reintroduction of native, but not a RING finger mutant, hPOSH restores virus release and Gag plasma membrane localization in hPOSH-depleted cells. Furthermore, expression of the RING finger mutant hPOSH inhibits virus release and induces accumulation of intracellular Gag in normal cells. Together, our results identify a previously undescribed step in HIV biogenesis and suggest a direct function for hPOSH-mediated ubiquitination in protein sorting at the trans-Golgi network. Consequently, hPOSH may be a useful host target for therapeutic intervention.protein sorting͞trafficking ͉ ubiquitin conjugation ͉ ubiquitin ligase ͉ HIV assembly ͉ HIV secretion

We have studied the direct interaction of the constitutive isoform of Hsp70 (Hsc70) with the DnaJ homolog, auxilin, a cofactor that binds to clathrin-coated vesicles and is required for their uncoating by Hsc70. Auxilin caused a 5-fold increase in Hsc70 ATPase activity and a corresponding increase in steady-state levels of bound ADP; the dissociation constant for this effect was 0.6 M. Auxilin also induced polymerization of Hsc70 and bound to the resulting polymer at a 1:1 molar ratio; here too the dissociation constant was 0.6 M. Both this binding and polymerization required ATP; the Hsc70 depolymerized with a 4-min half-life when ATP was completely hydrolyzed to ADP. Although auxilin induces polymerization stoichiometrically and other DnaJ homologs induce polymerization catalytically, these data show that auxilin is similar to other DnaJ homologs in its ability to activate the Hsc70 ATPase activity, to polymerize Hsc70, and in the nucleotide dependence of this polymerization. Furthermore, the 70-amino acid J-domain of auxilin polymerized Hsc70 with the same nucleotide dependence as intact auxilin. Therefore, although only auxilin and not other DnaJ homologs support uncoating, our data suggest that various DnaJ homologs share a common mechanism of interaction with Hsc70, perhaps because their J-domains interact similarly with Hsc70.

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrinbinding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrincoated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilinclathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.Many of the functions carried out by the Hsc70 (the constitutive isoform of Hsp70) class of molecular chaperones both in vivo and in vitro require the presence of J-domain proteins (1, 2). For example, Hsc70 alone cannot uncoat clathrin-coated vesicles in vitro (3); the J-domain protein auxilin must also be present in catalytic amounts (4). Furthermore, knockout of auxilin in yeast or knockdown of auxilin in Caenorhabditis elegans prevents endocytosis from taking place (5-7), showing that aside from Hsc70, auxilin is also required for uncoating to take place in these organisms.J-domain proteins are thought to function by inducing substrates to bind to Hsc70, and this has been directly demonstrated for auxilin by showing that auxilin is required for ATP-dependent binding of Hsc70 at pH 6, where there is no uncoating (8). Although the mechanism of action of J-domain proteins is not completely understood, it is generally thought that they first bind substrate and then transfer the substrate to Hsc70-ATP and, finally, induce one round of rapid ATP hydrolysis by Hsc70 (9, 10). This stabilizes the binding of substrate because most substrates detach rapidly from Hsc70-ATP but very slowly from Hsc70-ADP (11)(12)(13)(14).To carry out their function J-domain proteins must contain both a J-domain that interacts with Hsc70 and a substratebinding domain that interacts with substrate. In fact, mammalian auxilin, a multi-domain protein, consists of at least three domains, an N-terminal Pten-like domain (15) of unknown function, a clathrin-binding domain, and...

Several approaches were used to study the role of GroEL, the prototype chaperonin, in the nitrogen fixation (nij) system. An Escherichia coli groEL mutant transformed with the Klebsiella pneumoniae nif gene cluster accumulated very low to nondetectable levels of nitrogenase components compared with the isogenic wild-type strain or the mutant cotransformed with the wild-type groE operon. In K. pneumoniae, overexpression of the E. coli groE operon markedly accelerated the rate of appearance of the MoFe protein and its constituent polypeptides after the start of derepression. The groEL mutation in E. coli decreased NifA-dependent ,-galactosidase expression from the nifH promoter but did not affect the constitutive expression of nifA from the tet promoter or ntr-controlled expression from the nifLA promoter. The possibility that GroEL is required for the correct folding of NifA was supported by coimmunoprecipitation of NifA with anti-GroEL antibodies. Kinetic analyses of nitrogenase assembly in 35S pulse-chased K. pneumoniae pointed to the existence of high-molecular-weight intermediates in MoFe protein assembly and demonstrated the transient binding of newly synthesized NifH and NifDK to GroEL. Overall, these results indicate that GroEL fulfills both regulatory and structural functions in the nif system. Biological nitrogen fixation is carried out by dinitrogenase, a two-component enzyme complex. In molybdenum-dependent nitrogenases, the MoFe protein is responsible for substrate binding and reduction, whereas the Fe protein is the exclusive electron donor for the reaction. The MoFe protein is an OX212 tetramer of approximately 220 kDa assembled with four iron-sulfur clusters and containing two copies of an iron-molybdenum cofactor (FeMoco). The Fe protein is an ct2 dimer assembled with a single iron-sulfur cluster (reviewed in reference 38).In Klebsiella pneumoniae, the genetically best-characterized nitrogen-fixing organism, all the genetic information specifically required for nitrogen fixation resides within a contiguous 24-kb nif gene cluster (1). The 20 nif genes within this cluster are organized in seven or eight transcriptional units that together form a regulon, responding, via the ntr system, nifLA (28) and nijX (17), to the oxygen and fixednitrogen status of the cells. The nifA product serves as a nif-specific transcriptional activator, binding to upstream enhancer sequences of nif promoters (30) and acting in conjunction with RNA polymerase containing RpoN (U(54) as a sigma factor (reviewed in reference 28).The assembly of nitrogenase components is a complex process, involving the formation of oligomeric protein structures, the addition of metal clusters, and the biosynthesis of FeMoco and its association with the apo-MoFe protein. The underlying genetics are no less complex and, in K. pneumoniae, involve at least 10 nif genes. In addition to nifH and niJDK, encoding the structural subunits of the Fe protein (Kp2) and the MoFe protein (Kpl), respectively, this group includes genes involved in FeMoco syn...

The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically. The tertiary structure consists of six helices, where the first three are unique to auxilin and believed to be important in the catalytic uncoating of clathrin. The last three helices correspond to the canonical J-domain of Hsp40 proteins. The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure. Helix 1 is joined to helix 2 by a flexible linker. Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core. A long insert loop, also unique to the auxilin J-domain, is seen between helix 4 and helix 5. Comparison with a previously reported structure of auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain. The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein. In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen. The variety of biological functions performed by Hsp40 proteins such as auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.