A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction.
نویسندگان
چکیده
The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70-facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone.
منابع مشابه
Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals Roles for Hsc70 and Auxilin
We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxili...
متن کامل4SCIENTIFIC REVIEW Role of Auxilin and Heat Shock Protein 70kDa in Clathrin Uncoating
An interaction between auxilin and heat shock protein 70kDa (Hsc70) was initially discovered in 1995. There exists a large amount of data supporting the basis for their interaction in vitro and their function in clathrin uncoating in vivo. This review examines the key experiments in elucidating this interaction and introduces a third protein that may connect constriction or fission with hsc70/a...
متن کاملThe role of molecular chaperones in clathrin mediated vesicular trafficking
The discovery that the 70 kD "uncoating ATPase," which removes clathrin coats from vesicles after endocytosis, is the constitutively expressed Hsc70 chaperone was a surprise. Subsequent work, however, revealed that uncoating is an archetypal Hsp70 reaction: the cochaperone auxilin, which contains a clathrin binding domain and an Hsc70 binding J domain, recruits Hsc70(*)ATP to the coat and, conc...
متن کاملRecruitment 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 ...
متن کاملThe auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast
BACKGROUND In eukaryotic cells, clathrin-coated vesicles transport specific cargo from the plasma membrane and trans-Golgi network to the endosomal system. Removal of the clathrin coat in vitro requires the uncoating ATPase Hsc70 and its DnaJ cofactor auxilin. To date, a requirement for auxilin and Hsc70 in clathrin function in vivo has not been demonstrated. RESULTS The Saccharomyces cerevis...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Molecular biology of the cell
دوره 19 1 شماره
صفحات -
تاریخ انتشار 2008