Antibodies to cytoplasmic dynein heavy chain map the surface and inhibit motility¹

Abstract

Polyclonal antibodies have been raised against four 16 residue peptides with sequences taken from the C-terminal quarter of the human cytoplasmic dynein heavy chain. The sites are downstream from a known microtubule-binding domain associated with the “stalk” that protrudes from the motor domain. The antisera were assayed using bacterially expressed proteins with amino acid sequences taken from the human cytoplasmic dynein heavy chain. Every antiserum reacted specifically with the appropriate expressed protein and with pig brain cytoplasmic dynein, whether the protein molecules were denatured on Western blots or were in a folded state. But, whereas three of the four antisera recognized freshly purified cytoplasmic dynein, the fourth reacted only with dynein that had been allowed to denature a little. After affinity purification against the expressed domains, whole IgG molecules and Fab fragments were assayed for their effect on dynein activity in in vitro microtubule-sliding assays. Of the three anti-peptides that reacted with fresh dynein, one inhibited motility but the others did not. The way these peptides are exposed on the surface is compatible with a model whereby the dynein motor domain is constructed from a ring of AAA protein modules, with the C-terminal module positioned on the surface that interacts with microtubules. We have tentatively identified an additional AAA module in the dynein heavy chain sequence, which would be consistent with a heptameric ring.

Introduction

Cytoplasmic dynein (CD) is a multisubunit protein that uses ATP to translocate along microtubules (Holzbaur & Vallee, 1994). It has roles in cytoplasmic vesicle motility and, during mitosis, in chromosome separation and spindle organization Schroer 1994, Vallee and Sheetz 1996. Dynein purified from brain tissues is a homodimer of ∼500 kDa heavy chains (CDHCs) together with two intermediate chains (CDICs) of 70-80 kDa, four light intermediate chains (CDLICs) of 50-60 kDa and various light chains (CDLCs) (Holzbaur & Vallee, 1994). This complex drives microtubule sliding in vitro (Vallee & Sheetz, 1996). The dynactin protein complex that co-purifies with CD Gill et al 1991, Schroer and Sheetz 1991 seems to be needed for most of its in vivo activities (Karki & Holzbaur, 1999) but, in vitro, recombinant CDHC can by itself produce microtubule gliding with a velocity similar to that of native dynein (Mazumdar et al., 1996).

The N-terminal 1200-1300 residues dimerise as a stem domain that interacts with CDIC and tethers dynein to its cargo Habura et al 1999, Lyadurai et al 1999. The remaining ∼380 kDa form the globular catalytic head domain, responsible for direct interaction with microtubules, as well as for ATP hydrolysis and force production (Vallee, 1993). The central region contains four phosphate-binding loop (P-loop) motifs Gee and Vallee 1998, Vallee 1993, each part of a bona fide nucleotide-binding site (Mocz et al., 1998). However, ATPase activity is lost if the first P-loop is truncated or if the polypeptide is cleaved there by UV light in the presence of vanadate (Lee-Eiford et al., 1986). The head domain has been expressed without the stem in two systems Gee et al 1997, Koonce and Samso 1996, Koonce 1997 and shown to bind microtubules in an ATP-sensitive fashion and to undergo UV-vanadate cleavage. Any further truncation affects ATP binding or ATPase activity, though some microtubule binding persists if all four P-loops are removed or if ∼1170 residues are cut from the C terminus. A ∼300 residue region after the last P-loop includes two alpha-helical segments separated by a small globular domain. Gee & Vallee (1998) found that an expressed domain consisting only of this region was able to bind well to microtubules; in electron microscope images, shadowed specimens resembled the stalk that has been seen to extend from intact heads of both cytoplasmic and axonemal dyneins and to bind to axonemal microtubules in situ Goodenough and Heuser 1989, Burgess 1995. Experiments by Koonce (1997) support the existence of binding activity in this region but indicate that regions downstream of the stalk sequence may also have some affinity for microtubules.

A recent search of the sequence database (Neuwald et al., 1999) using a reference set of sequences in the AAA ATPase family Langer 2000, Patel and Latterich 1998 revealed the presence of six copies of the AAA module within the dynein motor domain sequence (see Figure 1), leading to the current model Vale 2000, King 2000 of the dynein motor domain as a ring of connected subunits. Each of the four P-loops is associated with an AAA module but the analysis (Neuwald et al., 1999) identified two additional modules in which P-loops are no longer recognizable as such. Here, we present evidence for a seventh module, also lacking a P-loop, which correlates with electron microscopic images that show each individual dynein head as a ring of at least seven globular lobes arranged around a central cavity (Samso et al., 1998). The modules without P-loops are highly conserved in sequence, despite having no known function. We have made antibodies to sequences that are found within the final two modules and are predicted to lie on an exposed surface. An investigation of their properties adds new details to the model of the motor domain and suggests that at least one of the AAA modules lacking a P-loop may be involved in interacting with microtubules.