Hooking She3p onto She2p for myosin-mediated cytoplasmic mRNA transport

Abstract

The segregation of approximately two dozen distinct mRNAs from yeast mother to daughter cell cytoplasm is a classical paradigm for eukaryotic mRNA transport. The information for transport resides in an mRNA element 40–100 nt in length, known as “zipcode.” Targeted transport requires properly positioned actin filaments and cooperative loading of mRNA cargo to myosin. Cargo loading to myosin uses myosin 4 protein (Myo4p), swi5p-dependent HO expression 2 protein (She2p) and 3 protein (She3p), and zipcode. We previously determined a crystal structure of Myo4p and She3p, their 1:2 stoichiometry and interactome; we furthermore showed that the motor complex assembly requires two Myo4p⋅She3p heterotrimers, one She2p tetramer, and at least a single zipcode to yield a stable complex of [Myo4p⋅She3p⋅She2p⋅zipcode] in 2:4:4:1 stoichiometry in vitro. Here, we report a structure at 2.8-Å resolution of a cocrystal of a She2p tetramer bound to a segment of She3p. In this crystal structure, the She3p segment forms a striking hook that binds to a shallow hydrophobic pocket on the surface of each She2p subunit of the tetramer. Both She3p hook and cognate She2p binding pocket are composed of highly conserved residues. We also discovered a highly conserved region of She3p upstream of its hook region. Because this region consists of basic and aromatic residues, it likely represents part of She3p’s binding activity for zipcode. Because She2p also exhibits zipcode-binding activity, we suggest that “hooking” She3p onto She2p aligns each of their zipcode-binding activities into a high-affinity site, thereby linking motor assembly to zipcode.

Localization elements and zip codes in the intracellular transport and localization of messenger RNAs in Saccharomyces cerevisiae

“…Structure‐function analysis of the ASH1 E3 element separately and in combination with its ligands, She2p and She3p using NMR and small angle X‐ray scattering (SARS) analysis revealed vital information about the molecular details of interaction between E3‐She2p‐She3p complex (Figure c) (Edelmann et al, ; Niessing, Hüttelmaier, Zenklusen, Singer, & Burley, ; N. Singh, Blobel, & Shi, ). Importantly, previous structural studies established that She2p forms an elongated tetrameric structure, which is crucial for the binding and function of this protein (M. Müller et al, ).…”

“…Two She2p dimers contact each other in a head-to-head fashion, leading to the formation of the tetramer. In the tetrameric state, the N termini of each subunit point to the poles of the elongated structure and the C termini point to the waist like the interface of the two opposite dimers (N. Singh, Blobel, et al, 2015). The waist like interface of She2p contains a lot of positively charged basic residues, which form the binding site of the E3 RNA element (M. Müller et al, 2009;Niessing et al, 2004).…”

“…The waist like interface of She2p contains a lot of positively charged basic residues, which form the binding site of the E3 RNA element (M. Müller et al, 2009;Niessing et al, 2004). Moreover, the co-crystal structure of a 30 residue long C-terminal domain of She3p, which interacts with She2p protein (termed as She3pep), showed that this segment contains a highly conserved LPVG motif that adopts a hook-like structure (N. Singh, Blobel, et al, 2015). Each hook contacts one of the four protomers of She2p through the hydrophobic pocket on the surface of She2p (N. Singh, Blobel, et al, 2015).…”

“…Moreover, the co-crystal structure of a 30 residue long C-terminal domain of She3p, which interacts with She2p protein (termed as She3pep), showed that this segment contains a highly conserved LPVG motif that adopts a hook-like structure (N. Singh, Blobel, et al, 2015). Each hook contacts one of the four protomers of She2p through the hydrophobic pocket on the surface of She2p (N. Singh, Blobel, et al, 2015). More recent structural studies revealed that the E3 RNA element alone adopts an elongated stem-loop structure with a highly flexible bulge in between the two helices (Edelmann et al, 2017).…”

“…Structure-function analysis of the ASH1 E3 element separately and in combination with its ligands, She2p and She3p using NMR and small angle X-ray scattering (SARS) analysis revealed vital information about the molecular details of interaction between E3-She2p-She3p complex (Figure 1c) (Edelmann et al, 2017;Niessing, Hüttelmaier, Zenklusen, Singer, & Burley, 2004;N. Singh, Blobel, & Shi, 2015).…”