Sherri L. Newmyer

Auxilin-dynamin interactions link the uncoating ATPase chaperone machinery with vesicle formation.

The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, after which the clathrin coat is removed by the chaperone Hsc70 and its cochaperone auxilin. Recent evidence suggests that the GTP-bound form of dynamin may recruit factors that execute the fission reaction. Here, we show that dynamin:GTP binds to Hsc70 and auxilin. We mapped two domains within auxilin that interact with dynamin, and these domains inhibit endocytosis when overexpressed in HeLa cells or when added in a permeable cell assay. The inhibition is not due to impairment of clathrin uncoating or to altered clathrin distribution in cells. Thus, in addition to its requirement for clathrin uncoating, our results show that auxilin also acts during the early steps of clathrin-coated vesicle formation. The data suggest that dynamin regulates the action of molecular chaperones in vesicle budding during endocytosis.

Rescue of His-42 → Ala Horseradish Peroxidase by a Phe-41 → His Mutation ENGINEERING OF A SURROGATE CATALYTIC HISTIDINE

Formation of the ferryl (FeIV=O) porphyrin radical cation known as Compound I in the reaction of horseradish peroxidase (HRP) with H2O2 is catalyzed by His-42, a residue that facilitates the binding of H2O2 to the iron and subsequent rupture of the dioxygen bond. An H42A mutation was shown earlier to decrease the rate of Compound I formation by a factor of ~106 and of guaiacol oxidation by a factor of ~104. In contrast, an F41A mutation has little effect on peroxidative catalysis (Newmyer, S. L., and Ortiz de Montellano, P. R. (1995) J. Biol. Chem. 270, 19430-19438). We report here construction, expression, and characterization of the F41H/H42A double mutant. The pH profile for guaiacol oxidation by this double mutant has a broad maximum at ~pH 6.3. Addition of H2O2 produces a Compound I species (λmax = 406 nm) that is reduced by 1 eq of K4Fe(CN)6 to the ferric state (λmax = 407 nm) without the detectable formation of Compound II. A fraction of the heme chromophore is lost in the process. The rate of Compound I formation for the F41H/H42A double mutant is 3.0 × 104 M−1 s−1. This is to be compared with 0.9 × 107 M−1 s−1 for wild-type HRP and 19 M−1 s−1 for the H42A mutant. The kcat values for guaiacol oxidation by wild-type, H42A, and F41H/H42A HRP are 300, 0.015, and 1.8 s−1. The corresponding kcat values for ABTS oxidation are 4900, 0.41, and 100 s−1, respectively. These results show that a histidine at position 41 substitutes, albeit imperfectly, for His-42 in peroxidative turnover of the enzyme. The F41H/H42A double mutant has peroxidative properties intermediate between those of the native enzyme and the H42A mutant. The F41H/H42A double mutant, however, is a considerably better thioanisole sulfoxidation and styrene epoxidation catalyst than native or H42A HRP. The surrogate catalytic residue introduced by the F41H mutation thus partially compensates for the H42A substitution used to increase access to the ferryl oxygen.

Physical and functional connection between auxilin and dynamin during endocytosis

During clathrin‐mediated endocytosis, the GTPase dynamin promotes formation of clathrin‐coated vesicles, but its mode of action is unresolved. We provide evidence that a switch in three functional states of dynamin (dimers, tetramers, rings/spirals) coordinates its GTPase cycle. Dimers exhibit negative cooperativity whereas tetramers exhibit positive cooperativity with respect to GTP. Our study identifies tetramers as the kinetically most stable GTP‐bound conformation of dynamin, which is required to promote further assembly into higher order structures such as rings or spirals. In addition, using fluorescence lifetime imaging microscopy, we show that interactions between dynamin and auxilin in cells are GTP‐, endocytosis‐ and tetramer‐dependent. Furthermore, we show that the cochaperone activity of auxilin is required for constriction of clathrin‐coated pits, the same early step in endocytosis known to be regulated by the lifetime of dynamin:GTP. Together, our findings support the model that the GTP‐bound conformation of dynamin tetramers stimulates formation of constricted coated pits at the plasma membrane by regulating the chaperone activity of hsc70/auxilin.

Assays and functional properties of auxilin-dynamin interactions.

The large GTPase dynamin is required for budding of clathrin-coated vesicles from the plasma membrane, but its mechanism of action is still not understood. Growing evidence indicates that the GTP-bound form of dynamin recruits downstream partners that execute the fission reaction. Recently, we reported nucleotide-dependent interactions between dynamin and auxilin, which suggested that auxilin cooperates with dynamin during vesicle formation. Here we describe three different in vitro assays that monitor auxilin-dynamin interactions, as well as fluorescence lifetime imaging microscopy that identify direct interactions between dynamin and auxilin in cells.