Class III myosins are motor proteins that contain an N-terminal kinase domain and a C-terminal actin-binding domain. We show that myosin IIIa, which has been implicated in nonsyndromic progressive hearing loss, is localized at stereocilia tips. Myosin IIIa progressively accumulates during stereocilia maturation in a thimble-like pattern around the stereocilia tip, distinct from the cap-like localization of myosin XVa and the shaft localization of myosin Ic. Overexpression of deletion mutants for functional domains of green fluorescent protein (GFP)-myosin IIIa shows that the motor domain, but not the actin-binding tail domain, is required for stereocilia tip localization. Deletion of the kinase domain produces stereocilia elongation and bulging of the stereocilia tips. The thimble-like localization and the influence myosin IIIa has on stereocilia shape reveal a previously unrecognized molecular compartment at the distal end of stereocilia, the site of actin polymerization as well as operation of the mechanoelectrical transduction apparatus.

Tobacco mosaic virus (TMV) induces the hypersensitive response (HR) in tobacco plants containing the N gene. This defence response is characterized by cell death at the site of virus infection and inhibition of viral replication and movement. A previous study indicated that a portion of the TMV replicase containing a putative helicase domain is involved in HR induction. Here, this observation is confirmed and extended by showing that non-viral expression of a 50 kDa TMV helicase fragment (p50) is sufficient to induce the N-mediated HR in tobacco. Like the HR elicited by TMV infection, transgenic expression of p50 induces a temperature-sensitive defence response. We demonstrate that recombinant p50 protein has ATPase activity, as suggested by the presence of conserved sequence motifs found in ATPase/helicase enzymes. A point mutation that alters one of these motifs abolishes ATPase activity in vitro but does not affect HR induction. These results suggest that features of the TMV helicase domain, independent of its enzymatic activity, are recognized by N-containing tobacco to induce TMV resistance.

Eukaryotic translation initiation factor 2 (eIF‐2) comprises three non‐identical subunits alpha, beta and gamma. In vitro, eIF‐2 binds the initiator methionyl‐tRNA in a GTP‐dependent fashion. Based on similarities between eukaryotic eIF‐2gamma proteins and eubacterial EF‐Tu proteins, we previously proposed a major role for the gamma‐subunit in binding guanine nucleotide and tRNA. We have tested this hypothesis by examining the biochemical activities of yeast eIF‐2 purified from wild‐type strains and strains harboring mutations in the eIF‐2gamma structural gene (GCD11) predicted to alter ligand binding by eIF‐2. The alteration of tyrosine 142 in yeast eIF‐2gamma, corresponding to histidine 66 in Escherichia coli EF‐Tu, dramatically reduced the affinity of eIF‐2 for Met‐tRNAi(Met) without affecting the k(off) value for guanine nucleotides. In contrast, non‐lethal substitutions at a conserved lysine residue (K250) in the putative guanine ring‐binding loop increased the off‐rate for GDP, thereby mimicking the function of the guanine nucleotide exchange factor eIF‐2B, without altering the apparent dissociation constant for Met‐tRNAi(Met). For eIF‐2[gamma‐K250R], the increased off‐rate also seen for GTP was masked by the presence of Met‐tRNAi(Met) in vitro. In vivo, increasing the dose of the yeast initiator tRNA gene suppressed the slow‐growth phenotype and reduced GCN4 expression in gcd11‐K250R and gcd11‐Y142H strains. These studies indicate that the gamma‐subunit of eIF‐2 does indeed provide EF‐Tu‐like function to the eIF‐2 complex, and further suggest that the level of Met‐tRNAi(Met) is critical for maintaining wild‐type rates of initiation in vivo.