Interactions of Cdc4p, a myosin light chain, with IQ-domain containing proteins in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe undergoes cell division through a medially placed actomyosin-based contractile ring. One of the key components of this ring is the F-actin based motor protein myosin II. The myosin II heavy chain Myo2p has two light-chain-binding domains, IQl and IQ2, which bind the essential light chain, Cdc4p, and the regulatory light chain, Rlc1p. Previously, we have reported the characterization of cells expressing Myo2p lacking the IQ2 domain that facilitates Myo2p interaction with Rlc1p. In this study, we have created and characterized S. pombe strains carrying precise deletions of IQ1 and the entire neck region encompassing the IQ1 and IQ2 domains. Surprisingly, we found that the entire neck region of Myo2p is dispensable for Myo2p function. Cells deleted for IQ1, IQ2 and the entire neck region of Myo2p do not display any obvious cytoskeletal abnormalities. Immunofluorescence studies indicated that Cdc4p localizes at the ring in early and late mitotic cells in a strain in which interactions of Cdc4p with both the myosin II heavy chains (Myo2p and Myp2p) are abolished. Unlike mutations in Rlc1p that are suppressed by a simultaneous deletion of its binding site on Myo2p, mutations in the essential light chain Cdc4p are not suppressed by deletion of its binding sites on Myo2p, suggesting that Cdc4p may have additional partners essential for cytokinesis. Consistent with this, we provide evidence that two other IQ-domain containing actomyosin ring proteins, Rng2p (an IQGAP-related protein) and Myo51p (a type V myosin heavy chain), physically interact with Cdc4p. We concluded that Cdc4p, a novel myosin light chain, interacts with multiple actomyosin ring components to effect cytokinesis.

Type II myosin regulatory light chain relieves auto-inhibition of myosin-heavy-chain function.

The F-actin based motor protein myosin II has a key role in cytokinesis. Here we show that the Schizosaccharomyces pombe regulatory light chain (RLC) protein Rlc1p binds to Myo2p in manner that is dependent on the IQ sequence motif (the RLC-binding site), and that Rlc1p is a component of the actomyosin ring. Rlc1p is important for cytokinesis at all growth temperatures and is essential for this process at lower temperatures. Interestingly, all deleterious phenotypes associated with the loss of Rlc1p function are suppressed by deletion of the RLC binding site on Myo2p. We conclude that the sole essential function of RLCs in fission yeast is to relieve the auto-inhibition of myosin II function, which is mediated by the RLC-binding site, on the myosin heavy chain (MHC).

Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis.

Cell division in many eukaryotes, including the fission yeast Schizosaccharomyces pombe, utilizes a contractile actomyosin ring. In S. pombe, the actomyosin ring is assembled at the medial cortex upon entry into mitosis and constricts at the end of anaphase to guide the centripetal deposition of the septum. Despite identification of several structural components essential for actomyosin ring assembly, the interdependencies between these gene-products in the process of ring assembly are unknown. This study investigates the role of Rng3p, a member of the UCS-domain containing protein family (Unc-45p, Cro1p, She4p), in actomyosin ring assembly. Null mutants in rng3 resemble deletion mutants in the type II myosin heavy chain (myo2) and rng3(ts) mutants show strong negative interactions with the myo2-E1 mutant, suggesting that Rng3p is involved in modulating aspects of type II myosin function. Interestingly, a green fluorescent protein (GFP) tagged Rng3p fusion is detected at the division site in the myo2-E1 mutant, but not in other myo2-alleles, wild-type cells or in 18 other cytokinesis mutants. Assembly and maintenance of Rng3p at the division site in the myo2-E1 mutant requires F-actin. Rng3p is also required for the proper assembly of Myo2p and F-actin into a functional actomyosin ring but is not necessary for their accumulation at the division site. We conclude that Rng3p is a novel component of the F-actin cytoskeleton essential for a late step in actomyosin ring assembly and that it might monitor some aspect of type II myosin assembly during actomyosin ring construction.

Identification of a 26S proteasome-associated UCH in fission yeast.

We have identified a 26S proteasome-associated ubiquitin carboxyl-terminal hydrolase (UCH) in Schizosaccharomyces pombe. The gene (designated uch2+) encodes a protein containing a UCH catalytic domain at its N-terminus and a short extension at its C-terminus. uch2+ is nonessential as the uch2 null mutant strain showed no significant difference from the wild-type strain. The GFP-tagged Uch2p is localized predominantly to the nuclear periphery, which is similar to the 26S proteasome localization. Deletion of the C-terminal extension of Uch2p resulted in a drastic change of its subcellular localization: it showed a generally diffused distribution instead of a perinuclear pattern. Glycerol gradient centrifugation analysis and coimmunoprecipitation studies of fission yeast extracts using anti-Mts4p antiserum suggest that Uch2p is associated with the 26S proteasome and the association of Uch2p with the 26S proteasome is mediated by its C-terminal extension.

Evidence for F-actin-dependent and -independent mechanisms involved in assembly and stability of the medial actomyosin ring in fission yeast.

Cell division in a number of eukaryotes, including the fission yeast Schizosaccharomyces pombe, is achieved through a medially placed actomyosin-based contractile ring. Although several components of the actomyosin ring have been identified, the mechanisms regulating ring assembly are still not understood. Here, we show by biochemical and mutational studies that the S.pombe actomyosin ring component Cdc4p is a light chain associated with Myo2p, a myosin II heavy chain. Localization of Myo2p to the medial ring depended on Cdc4p function, whereas localization of Cdc4p at the division site was independent of Myo2p. Interestingly, the actin-binding and motor domains of Myo2p are not required for its accumulation at the division site although the motor activity of Myo2p is essential for assembly of a normal actomyosin ring. The initial assembly of Myo2p and Cdc4p at the division site requires a functional F-actin cytoskeleton. Once established, however, F-actin is not required for the maintenance of Cdc4p and Myo2p medial rings, suggesting that the attachment of Cdc4p and Myo2p to the division site involves proteins other than actin itself.

Isolation and characterization of new fission yeast cytokinesis mutants.

Schizosaccharomyces pombe is an excellent organism in which to study cytokinesis as it divides by medial fission using an F-actin contractile ring. To enhance our understanding of the cell division process, a large genetic screen was carried out in which 17 genetic loci essential for cytokinesis were identified, 5 of which are novel. Mutants identifying three genes, rng3(+), rng4(+), and rng5(+), were defective in organizing an actin contractile ring. Four mutants defective in septum deposition, septum initiation defective (sid)1, sid2, sid3, and sid4, were also identified and characterized. Genetic analyses revealed that the sid mutants display strong negative interactions with the previously described septation mutants cdc7-24, cdc11-123, and cdc14-118. The rng5(+), sid2(+), and sid3(+) genes were cloned and shown to encode Myo2p (a myosin heavy chain), a protein kinase related to budding yeast Dbf2p, and Spg1p, a GTP binding protein that is a member of the ras superfamily of GTPases, respectively. The ability of Spg1p to promote septum formation from any point in the cell cycle depends on the activity of Sid4p. In addition, we have characterized a phenotype that has not been described previously in cytokinesis mutants, namely the failure to reorganize actin patches to the medial region of the cell in preparation for septum formation.

Rng2p, a protein required for cytokinesis in fission yeast, is a component of the actomyosin ring and the spindle pole body.

BACKGROUND: An actomyosin-based contractile ring plays a pivotal role in cytokinesis. Despite the identification of many components of the ring, the steps involved in its assembly are unknown. The fission yeast Schizosaccharomyces pombe is an attractive organism in which to study cytokinesis because its cell cycle has been well characterized; it divides by medial fission using an actomyosin ring; and a number of S. pombe mutants defective in actomyosin ring assembly have been isolated. Here, we have characterized one such mutant, rng2. RESULTS: Temperature-sensitive rng2 mutants accumulated F-actin cables in the medial region of the cell but failed to organize the cables into a ring. In rng2-null mutants, only a spot-like structure containing F-actin was detected. The rng2+ gene encodes a protein related to human IQGAP1, a protein that binds actin and calmodulin and is a potential effector for the Rho family of GTPases. Rng2p localized to the actomyosin ring and to the spindle pole body (SPB) of interphase and mitotic cells. Localization of Rng2p to the actomyosin ring but not the SPB required F-actin. Rng2p interacted with calmodulin, a component of the SPB and the actomyosin ring. The rng2 gene showed genetic interactions with three other actomyosin ring assembly mutants, cdc4, cdc12, and rng5. CONCLUSIONS: The S. pombe IQGAP-related protein Rng2p is a component of the actomyosin ring and the SPB and is required for actomyosin ring construction following assembly of F-actin at the division site.

Development of a sequence characterized amplified region (SCAR) based indirect selection method for a dominant blast-resistance gene in rice.

Two randomly amplified polymorphic DNA (RAPD) markers, OPF6(2700) and OPH18(2400), tightly linked to Pi-10, a dominant blast-resistance gene conferring complete resistance to isolate 106 (international race IB46) of the blast fungus were identified. To derive sequence characterized amplified regions (SCARs) from OPF6(2700) and OPH18(2400), these amplified RAPD products were cloned and sequenced. Nucleotide sequence information, obtained for each end of the two linked RAPD markers, was used to design 24-mer oligonucleotide primers for PCR amplification of the respective SCARs. Polymorphisms appearing as differences in the length of the SCAR's alternate alleles were considered for the indirect selection of Pi-10. Such polymorphisms converted the linked dominant RAPD loci into codominant SCAR markers and also facilitated the indirect scoring of the blast-resistant and blast-susceptible genotypes. The development of length variant codominant SCAR markers linked to a major gene for blast resistance in rice is described. The codominant SCARs will facilitate marker-assisted selection of the Pi-10 locus in rice breeding programs and will also be useful as genetic markers for high resolution mapping of the Pi-10 region.