Clathrin-coated vesicles transport selected integral membrane proteins from the cell surface and the trans-Golgi network to the endosomal system. Before fusing with their target the vesicles must be stripped of their coats. This process is effected by the chaperone protein hsp70c together with a 100K cofactor which we here identify as the coat protein auxilin. Auxilin binds with high affinity to assembled clathrin lattices and, in the presence of ATP, recruits hsp70c. Dissociation of the lattice does not depend as previously supposed on clathrin light chains or on the amino-terminal domain of the heavy chain. The presence of a J-domain at its carboxy terminus now defines auxilin as a member of the DnaJ protein family. In conjunction with hsp70, DnaJ proteins catalyse protein folding, protein transport across membranes and the selective disruption of protein-protein interactions. We show that deletion of the J-domain of auxilin results in the loss of cofactor activity.

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Crossbridge models of muscle contraction based on biochemical studies predict that there may be a relationship between the rate-limiting step in the actomyosin ATPase cycle in vitro and the rate of force development in vivo. In the present study, we measured the rate of force redevelopment in skinned rabbit muscle fibers following unloaded isotonic shortening and a rapid restretch. For comparison, ATPase activity was measured under identical conditions, using myosin subfragment-1 chemically crosslinked to actin. We found that the time course of force redevelopment is well fitted by a single exponential function, implying that force redevelopment is a first-order process, described by a single rate constant. The magnitude of this rate constant is in close agreement with the rate constant necessary to simulate the experimental force-velocity relation on the basis of a crossbridge model of the type proposed by A. F. Huxley in 1957. In addition, the observed close correlation between the rate constant for force redevelopment and the maximal actinactivated actomyosin ATPase rate under a variety of conditions suggests that the step that determines the rate of force generation in the crossbridge cycle may be the physiological equivalent of the rate-limiting step in the actomyosin ATPase cycle in solution.It is now widely accepted that skeletal muscle contraction occurs when the thin (actin) filaments slide past the thick (myosin) filaments. This process is driven by crossbridges that extend from the myosin filaments and cyclically interact with the actin filaments as ATP is hydrolyzed. This cyclic interaction causes maximal force development when the muscle is prevented from shortening (isometric contraction) and maximal shortening velocity when the actin and myosin filaments are allowed to slide freely past each other (unloaded isotonic contraction).In 1957, A. F. Huxley (1) presented the first quantitative model of crossbridge action. In this model he suggested that crossbridges have a moderate rate of attachment (f) to actin to form the force-generating state, a very slow rate of detachment (gl) during their work-stroke while they are exerting force, and a quite rapid rate of detachment (g2) at the end of their work-stroke. This model was able to account quantitatively for the relationship between force and shortening velocity exhibited by the muscle (force-velocity curve) and also for the leveling offof energy utilization by the muscle at high velocity. However, it is not a unique model; a model with a very different set of values forfand g was also able to account for these physiological data (2).Since these crossbridge models were proposed, considerable progress has been made in understanding the biochemistry of muscle contraction (3, 4). Studies on the interaction of actin, myosin, and ATP have shown that myosin can occur in two major conformations (5). With ATP or ADP'Pi bound, myosin occurs in a weak-binding conformation that binds very weakly to actin and attaches to and detaches from actin very rapid...

Recent experimental data on the equilibrium binding of myosin subfragment 1 (S-1) to regulated actin filaments in the presence and in the absence of Ca2+ are analyzed by using In a recent paper, Greene and Eisenberg (1) presented experimental data on the equilibrium binding of the myosin-subfragment-l-ADP complex (hereafter simply referred to as S-1, for brevity) to the troponin-tropomyosin-F-actin complex (regulated F-actin), in the presence and in the absence of Ca2+. The binding isotherms show interesting cooperativity (the data are included in Fig. 2, below). A tentative interpretation and analysis of the data were given (1) based on a simple model (ref.2, equations 10-12a with T = 0; ref. 3, equations 7-70 and 7-71 with r = 0) that does not include nearest-neighbor cooperativity in a quantitative way (see the discussion of Eqs. 22-25, below). Essentially the same model (in the T = 0 case), with equivalent equations, was used later by Monod et al. (4) to account for the allosteric behavior of regulatory proteins. It is the purpose of the present paper to reinterpret the same data in terms of a more refined model that includes nearest-neighbor interactions between troponin-tropomyosin units on the F-actin.We are extending the approach of the present paper to the in vitro and in vivo steady-state ATPase activity of regulated actomysin; this steady-state system serves as a good illustration of recent general theoretical studies (5-9) on the effect of nearest-neighbor interactions on steady-state enzyme behavior.

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.