Signal sequence-independent targeting of MID2 mRNA to the endoplasmic reticulum by the yeast RNA-binding protein Khd1p.

Localization of mRNAs depends on specific RNA-binding proteins (RBPs) and critically contributes not only to cell polarization but also to basal cell function. The yeast RBP Khd1p binds to several hundred mRNAs, the majority of which encodes secreted or membrane proteins. We demonstrate that a subfraction of Khd1p associates with artificial liposomes and endoplasmic reticulum (ER), and that Khd1p endomembrane association is partially dependent on its binding to RNA. ER targeting of at least two mRNAs, MID2 and SLG1/WSC1, requires KHD1 but is independent of their translation. Together, our results suggest interdependence of Khd1p and mRNA for their targeting to the ER and presents additional evidence for signal sequence-independent, RBP-mediated mRNA targeting.

The structure of the Myo4p globular tail and its function in ASH1 mRNA localization.

Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain.

Asymmetric localization of the adaptor protein Miranda in neuroblasts is achieved by diffusion and sequential interaction of Myosin II and VI.

The adaptor protein Miranda plays a pivotal role in the asymmetric cell division of neuroblasts by asymmetrically segregating key differentiation factors. Miranda localization requires Myosin VI and Myosin II. The apical-then-basal localization pattern of Miranda detected in fixed tissue, and the localization defects in embryos lacking Myosin VI, suggest that Miranda is transported to the basal pole as a Myosin VI cargo. However, the mode and temporal sequence of Miranda localization have not been characterized in live embryos. Furthermore, it is unknown whether Miranda and PON, a second adaptor protein required for asymmetric protein localization, are both regulated by Myosin II. By combining immunofluorescence studies with time-lapse confocal microscopy, we show that Miranda protein forms an apical crescent at interphase, but is ubiquitously localized at prophase in a Myosin-II-dependent manner. FRAP analysis revealed that Miranda protein reaches the basal cortex by passive diffusion throughout the cell, rather than by long-range Myosin VI-directed transport. Myosin VI acts downstream of Myosin II in the same pathway to deliver diffusing Miranda to the basal cortex. PON localization occurs mainly along the cortex and requires Myosin II but not Myosin VI, suggesting that distinct mechanisms are employed to localize different adaptor proteins during asymmetric cell division.