A recent study showed that the globular tail of Myo4p is not required for the localization of GFP-MS2–tethered particles to the bud and for the inheritance of ER (Bookwalter et al., 2009
). This observation suggested that the globular tail might be dispensable for the localization of endogenous ASH1
mRNA and thus also for inhibition of mating type switching in the daughter cell. It further raised the question of whether the globular tail of Myo4p has any function. Here, we used an experimentally refined, globular tail–lacking Myo4p to confirm the previous findings from Bookwalter et al. (2009)
(Fig. S1, A–F).
However, when analyzing mother cell–specific expression of the HO endonuclease in cells expressing a globular tail–lacking Myo4p fragment, we found that this process is impaired (). The subsequent analysis of ASH1
mRNA localization by in situ hybridization and of Myo4p localization by immunofluorescence staining consistently showed that the globular tail is required for full Myo4p activity in vivo (). In vitro interaction studies with different Myo4p fragments and its adapter She3p also demonstrated that the globular tail is required for efficient complex formation (). Analysis of surface features of the Myo4p globular tail and subsequent mutational studies identified a set of mutations in the globular tail that impairs She3p binding in vitro and reduces ASH1
mRNA localization in vivo ( and Fig. S5, A–D). Furthermore, the protease-sensitive linker between the coiled-coil region and the globular tail contains a hydrophobic sequence patch that, upon mutation, also results in impaired She3p binding and reduced ASH1
mRNA localization ( and ). Thus, deletion studies as well as point mutations in the linker region and in the globular tail confirm the requirement of both regions for She3p binding and Myo4p function. An obvious question arising from these findings is why two independent groups (this study and Bookwalter et al., 2009
) were unable to detect this localization defect by analyzing RNA localization with GFP-MS2–tethered particles.
It has been reported that insertion of MS2-stem loops into the 3′ UTR of ASH1
mRNA and the tethering of multiple GFP-MS2 molecules to the 3′ UTR reduces the number of ASH1
transport particles and increases their size (Bertrand et al., 1998
; Lange et al., 2008
). This effect is particularly pronounced when the reporter RNA is expressed from a strong GAL1
promoter. In the majority of cases, we detected only a single large particle per cell (see Fig. S1 D), whereas normal cells contain several ASH1
-mRNA particles, even when ASH1
mRNA is overexpressed (Lange et al. 2008
). In light of these considerations, our findings indicate that GFP-MS2–tethered particles may not always faithfully recapitulate endogenous ASH1
mRNA localization. This technical limitation might be particularly true for defects that do not result in a total loss of ASH1
mRNA localization, like the deletion of the globular tail studied here.
For ER inheritance, moderately impaired Myo4p-dependent transport may also not result in detectable differences. Certain aspects of Myo4p function differ for ASH1
mRNA localization function. For ASH1
mRNA localization, the motor Myo4p, full-length She3p, the RNA-binding protein She2p, as well as a number of additional RNA-binding factors are required; in contrast, Myo4p and the N-terminal domain of She3p are sufficient for ER inheritance (Estrada et al., 2003
; Schmid et al. 2006
). In addition, cortical ER is tethered to the Myo4p–She3p complex by an unknown mechanism and its inheritance does not seem to require an anchoring step at the bud tip. It has also been disputed how important the contribution of Myo4p to ER inheritance is (Reinke et al., 2004
). Finally, it should be noted that ER inheritance is happening early in the cell cycle, before ASH1
mRNA is expressed and localized. In summary, there are several differences between ER inheritance and ASH1
mRNA localization that could explain the lacking defect in ER inheritance. More molecular details of ER inheritance may be required to understand the mechanistic basis of this difference.
The x-ray structure of the Myo4p globular tail revealed an almost entirely α-helical domain with a hook-like arrangement. The sequence identity between the globular tails of Myo4p and Myo2p is only 25% (Fig. S2). It is therefore remarkable that both domains share a very similar structural arrangement, composed of two almost identical subdomains. Considering the very different functions of Myo2p and Myo4p, and the low sequence identity for their globular tails, it is possible that similar domain architectures are also present in globular tail domains of other type V myosins. The extremely low surface conservation indicates that these domains evolved significantly diverging surface properties to allow for the binding of very different cargoes or cargo adapters.
However, when superposing the globular tails of Myo4p and Myo2p, we found that the surface region of Myo4p required for She3p binding (Fig. S5, B and C) overlaps with residues in Myo2p important for peroxisome inheritance and interaction with its peroxisome cargo adapter Inp2p. (Fig. S5, E and F; Fagarasanu et al., 2009
). This surface is also required for the interaction of Myo2p with the Rab GTPases Ypt31/32 and motility of endocytic compartments (Lipatova et al., 2008
). These structural overlaps suggest that at least some type V myosins might have a common functional site in their globular tail, albeit with different cargo specificities.
Our structural analyses also revealed that the Myo4p globular tail lacks residues conserved in other MyoV motors (; Figs. S2 and S4) that are required for auto-inhibition of their motor domains. The lacking conservation of this surface area suggests that Myo4p does not undergo auto-inhibition by the previously described mechanism (Li et al., 2008
). Because Myo4p is strictly monomeric in absence of cargo complexes (Dunn et al., 2007
; Heuck et al., 2007
; Hodges et al., 2008
) and may require oligomerization for processive movement (Dunn et al., 2007
; Heuck et al., 2007
), such an auto-inhibition mechanism might indeed not be required.
Database searches yielded strong structural similarity of the globular tails of Myo4p and Myo2p to components of the membrane-tethering exocyst, Dsl1 and COG complexes (Fig. S3, D–I). The role of these factors in membrane tethering and the localization of exocyst components at the bud tip in yeast (Ungar et al., 2006
; Wu et al., 2008
; He and Guo, 2009
) suggest that MyoV globular tails could potentially also tether to membrane sites.
All exocyst components with known structures share a similar overall fold (Munson and Novick, 2006
). Because these exocyst components are thought to interact with each other through their elongated helical bundles (Dong et al., 2005
), a similar interaction with exocyst components could also be envisioned for the structurally related MyoV globular tails. However, immunoprecipitation experiments with the globular tail of Myo4p failed to yield an interaction with the exocyst components Sec3p, Sec5p, Sec6p, Sec10p, Sec15p, Exo70p, or Exo84p above background levels (unpublished data). Because interaction studies with membrane-associated complexes are often technically demanding, it might be that experimental limitations prevented us from detecting binding to the exocyst complex. More thorough experiments will be required to rigorously assess the functional relationship of exocyst components and the Myo4p globular tail.
Regardless of this preliminary result, it will be interesting to see if globular tails of other MyoV motors can interact with exocyst components. The most pronounced difference between the Myo2p and Myo4p globular tails is the relative arrangement of their subdomains I and II and their resulting different overall shapes. In case a subset of MyoV globular tails indeed binds to exocyst components, such a difference in subdomain orientation could influence their propensity to interact with the elongated helical bundles of the exocyst complex.
In type V myosins of higher eukaryotes, regions outside the globular tail also contribute to binding of cargo complexes (Li and Nebenführ, 2008
). For instance, vertebrate Myo5a interacts with its adapter melanophilin through the globular tail and a more N-terminal motif in the rod region (Wu et al., 2002
). A second example is Myo5b, where also a motif in the rod and in the globular tail has been suggested to mediate binding to its Rab11–FIP2 cargo complex (Lapierre et al., 2001
). These binding motifs outside the globular tail are only found in alternatively spliced, tissue-specific versions of Myo5a/b, whereas their other splice forms bind to different cargo complexes (Li and Nebenführ, 2008
). In yeast, Myo2p and Myo4p exist only as a single isoform. Based on sequence identity (Fig. S2 B), dimerization state (Dunn et al., 2007
; Heuck et al., 2007
; Hodges et al., 2008
), and the presence of an auto-inhibition motif in the globular tail (), Myo2p is arguably the closer homologue to MyoV from vertebrates. However, only Myo4p binds its cargo in a way reminiscent of the alternatively spliced Myo5a and Myo5b motors. In summary, we find that the generally assumed requirement of the globular tail in type V myosins for cargo binding also holds true for Myo4p from yeast.