All class-I myosins have a single heavy chain consisting of an N-terminal globular head with actin-activated ATPase activity, an IQ domain which binds one or more light chains, and a C-terminal non-helical tail with a basic region adjacent to the motor domain. In addition, long-tail Acanthamoeba and Dictyostelium class-I myosins have a GPA (Acanthamoeba) or GPQ (Dictyostelium) region and an SH3 region following the basic region. Mammalian Myo1C and Myo1G bind to acidic phospholipids in vitro and in vivo through a putative PH domain within the basic region that may bind specifically to PIP2. Although Acanthamoeba myosin IC (AMIC) contains a putative PH domain within the basic region, we showed previously that AMIC shows no specificity for binding to PIP2 in vitro. AMIC binds to phospholipid vesicles containing either phosphatidylserine, PIP2 or both in proportion to their net negative charge irrespective of their phospholipid composition. Moreover, presumably because of the high negative charge of PIP2 and PIP3, endogenous AMIC colocalizes with PIP2/PIP3 in the Acanthamoeba plasma membrane. The basis of the affinity of AMIC for acidic phospholipid in vitro is a short sequence (13 residues) enriched with basic and hydrophobic amino acids (the BH-site) that lies within the putative PH domain. In vitro studies with synthetic peptides and sequence analysis by a novel computer program identified BH-sites in many class I myosins, including Dictyostelium myosin IB (DMIB), and also non-myosin proteins, suggesting that plasma membrane-association of proteins through non-specific BH-sites may be wide spread. Recently, lipid/membrane binding of mammalian Myo1E was shown by the Ostap laboratory to be more similar to the binding of AMIC than the binding of mammalian Myo1C. The colocalization of endogenous AMIC and PIP2/PIP3 in the plasma membrane of Acanthamoeba is consistent with, but does not prove, an important role for the BH-site. One needs to be able to express labeled wild-type and mutant constructs to determine the importance of the BH-site and if other factors might also be involved in membrane localization in live cells. Therefore, we chose to work with Dictyostelium for which all of the necessary tools are available. When placed in non-nutrient medium, Dictyostelium amoebae chemotax towards aggregation centers initiated by cells secreting cAMP. Chemotaxing cells elongate and polarize, with some proteins moving to the front and others to the rear, and secrete cAMP which attracts neighboring cells thus forming streams of chemotaxing amoebae. DMIB has been shown by us and others to concentrate at the plasma membrane in vegetative cells, in the cytoplasm at the front of motile amoebae and at sites of cell-cell contact. We asked if the BH-site is required for the association of DMIB with the plasma membrane, if DMIB shows preference for PIP2/PIP3-enriched regions of the plasma membrane, and what factors, in addition to the BH-site, might be required for the dynamic relocalization of DMIB in starved, motile and chemotaxing amoebae. First we confirmed by antibody-staining of fixed cells that endogenous DMIB is localized uniformly on the plasma membrane of resting cells, at active protrusions and cell-cell contacts of randomly moving cells, and at the front of motile polarized cells. By expression of eGFP-DMIB and eGFP-labeled mutants in DMIB-null cells, we found that the BH-site, residues 801KKKVLVHTLIRR812, is required for association of DMIB with the plasma membrane at all stages. Mutants with a deleted BH-site or with hydrophobic amino acids in the BH-site replaced by alanines did not localize to the plasma membrane. Moreover, the basic region of the tail (that contains the BH-site is sufficient for localization to the plasma membrane as expressed constructs missing head and IQ domains or the GPQ and SH3 domains also localized to the plasma membrane. All the membrane regions enriched in DMIB were also enriched in either or both PIP2 and PIP3. Thus, the charge-based specificity of the BH-site allows for in vivo specificity of DMIB for PIP2/PIP3 that is functionally similar but mechanistically different from the steric specificity of other class-I myosins based on PH domains. However, although required for plasma membrane association, the BH-site alone is not sufficient for proper relocalization of DMIB during random cell movement and starvation-induced cell polarization since localization of tail alone was different than localization of full length myosin. In freshly plated cells, both DMIB and Tail localized uniformly to the plasma membrane but the fraction of cytoplasmic DMIB was higher than that of Tail. In randomly moving cells, both DMIB and Tail were enriched in protrusions and cell-cell contacts but Tail was also present at the remaining regions of the plasma membrane from which DMIB was absent. The most striking difference between DMIB and Tail was that upon starvation DMIB relocated to the front of elongated cells whereas Tail remained relatively evenly distributed on the entire plasma membrane with some enrichment at the rear. The differences between Tail and DMIB localization could be explained by the presence of a cytoplasmic factor that interacts with the myosin head, pulling myosin off the membrane into the cytoplasm. Such a factor was proposed earlier by the Titus laboratory based on fractionation studies but never identified. Now we have shown that the cytoplasmic factor(s) that keeps full-length myosin off the membrane involves F-actin. The localization of DMIB-E407K, in which binding to F-actin through the head is compromised, was intermediate between localization of DMIB and the localization of Tail. Also, other than Tail and Head alone (and mutants with a deleted or non-functional BH-site), DMIB-E407K was the only construct whose relocalization was seriously handicapped compared to DMIB. Therefore, at least in all situations that we have studied, the two main factors determining DMIB localization are binding to the plasma membrane (lipids) through the BH-site in the tail and binding to cytoplasmic F-actin through the ATP-sensitive actin-binding site in the head. These two major interactions competing with each can explain most of the differences between the localization of expressed tail and full-length DMIB.
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