Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.     

AvrBsT Acetylates Arabidopsis ACIP1, a Protein that Associates with Microtubules and Is Required for Immunity

Bacterial pathogens of plant and animals share a homologous group of virulence factors, referred to as the YopJ effector family, which are translocated by the type III secretion (T3S) system into host cells during infection. Recent work indicates that some of these effectors encode acetyltransferases that suppress host immunity. The YopJ-like protein AvrBsT is known to activate effector-triggered immunity (ETI) in Arabidopsis thaliana Pi-0 plants; however, the nature of its enzymatic activity and host target(s) has remained elusive. Here we report that AvrBsT possesses acetyltransferase activity and acetylates ACIP1 (for ACETYLATED INTERACTING PROTEIN1), an unknown protein from Arabidopsis. Genetic studies revealed that Arabidopsis ACIP family members are required for both pathogen-associated molecular pattern (PAMP)-triggered immunity and AvrBsT-triggered ETI during Pseudomonas syringae pathovar tomato DC3000 (Pst DC3000) infection. Microscopy studies revealed that ACIP1 is associated with punctae on the cell cortex and some of these punctae co-localize with microtubules. These structures were dramatically altered during infection. Pst DC3000 or Pst DC3000 AvrRpt2 infection triggered the formation of numerous, small ACIP1 punctae and rods. By contrast, Pst DC3000 AvrBsT infection primarily triggered the formation of large GFP-ACIP1 aggregates, in an acetyltransferase-dependent manner. Our data reveal that members of the ACIP family are new components of the defense machinery required for anti-bacterial immunity. They also suggest that AvrBsT-dependent acetylation in planta alters ACIP1''s defense function, which is linked to the activation of ETI.     

Myosin 1G (Myo1G) is a haematopoietic specific myosin that localises to the plasma membrane and regulates cell elasticity

Immune cells navigate through different environments where they experience different mechanical forces. Responses to external forces are determined by the mechanical properties of a cell and they depend to a large extent on the actin-rich cell cortex. We report here that Myo1G, a previously uncharacterised member of class I myosins, is expressed specifically in haematopoietic tissues and cells. It is associated with the plasma membrane. This association is dependent on a conserved PH-domain-like myosin I tail homology motif and the head domain. However, the head domain does not need to be a functional motor. Knockdown of Myo1G in Jurkat cells decreased cell elasticity significantly. We propose that Myo1G regulates cell elasticity by deformations of the actin network at the cell cortex.

Structured summary

MINT-7307273: MYO1G (uniprotkb:B0I1T2) and Actin (uniprotkb:P60709) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7307283: TfR (uniprotkb:P02786) and MYO1G (uniprotkb:B0I1T2) colocalize (MI:0403) by cosedimentation through density gradients (MI:0029)     

A Specific Light Chain of Kinesin Associates with Mitochondria in Cultured Cells

The motor protein kinesin is implicated in the intracellular transport of organelles along microtubules. Kinesin light chains (KLCs) have been suggested to mediate the selective binding of kinesin to its cargo. To test this hypothesis, we isolated KLC cDNA clones from a CHO-K1 expression library. Using sequence analysis, they were found to encode five distinct isoforms of KLCs. The primary region of variability lies at the carboxyl termini, which were identical or highly homologous to carboxyl-terminal regions of rat KLC B and C, human KLCs, sea urchin KLC isoforms 1–3, and squid KLCs. To examine whether the KLC isoforms associate with different cytoplasmic organelles, we made an antibody specific for a 10-amino acid sequence unique to B and C isoforms. In an indirect immunofluorescence assay, this antibody specifically labeled mitochondria in cultured CV-1 cells and human skin fibroblasts. On Western blots of total cell homogenates, it recognized a single KLC isoform, which copurified with mitochondria. Taken together, these data indicate a specific association of a particular KLC (B type) with mitochondria, revealing that different KLC isoforms can target kinesin to different cargoes.     

The Class V Myosin Myo2p Is Required for Fus2p Transport and Actin Polarization during the Yeast Mating Response

Mating yeast cells remove their cell walls and fuse their plasma membranes in a spatially restricted cell contact region. Cell wall removal is dependent on Fus2p, an amphiphysin-associated Rho-GEF homolog. As mating cells polarize, Fus2p-GFP localizes to the tip of the mating projection, where cell fusion will occur, and to cytoplasmic puncta, which show rapid movement toward the tip. Movement requires polymerized actin, whereas tip localization is dependent on both actin and a membrane protein, Fus1p. Here, we show that Fus2p-GFP movement is specifically dependent on Myo2p, a type V myosin, and not on Myo4p, another type V myosin, or Myo3p and Myo5p, type I myosins. Fus2p-GFP tip localization and actin polarization in shmoos are also dependent on Myo2p. A temperature-sensitive tropomyosin mutation and Myo2p alleles that specifically disrupt vesicle binding caused rapid loss of actin patch organization, indicating that transport is required to maintain actin polarity. Mutant shmoos lost actin polarity more rapidly than mitotic cells, suggesting that the maintenance of cell polarity in shmoos is more sensitive to perturbation. The different velocities, differential sensitivity to mutation and lack of colocalization suggest that Fus2p and Sec4p, another Myo2p cargo associated with exocytotic vesicles, reside predominantly on different cellular organelles.     

Structural Insights into the Globular Tails of the Human Type V Myosins Myo5a,Myo5b,and Myo5c

Vertebrate type V myosins (MyoV) Myo5a, Myo5b, and Myo5c mediate transport of several different cargoes. All MyoV paralogs bind to cargo complexes mainly by their C-terminal globular domains. In absence of cargo, the globular domain of Myo5a inhibits its motor domain. Here, we report low-resolution SAXS models for the globular domains from human Myo5a, Myo5b, and Myo5c, which suggest very similar overall shapes of all three paralogs. We determined the crystal structures of globular domains from Myo5a and Myo5b, and provide a homology model for human Myo5c. When we docked the Myo5a crystal structure into a previously published electron microscopy density of the autoinhibited full-length Myo5a, only one domain orientation resulted in a good fit. This structural arrangement suggests the participation of additional region of the globular domain in autoinhibition. Quantification of the interaction of the Myo5a globular domain with its motor complex revealed a tight binding with dissociation half-life in the order of minutes, suggesting a rather slow transition between the active and inactive states.     

Nonmuscle Myosin Heavy Chain B Is Recognized by a Monoclonal Antibody that Inhibits GM1-Enhanced Neuritogenesis

Abstract: Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1-binding proteins and shown to inhibit GM1-enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.     

Myosin Vb Is Associated with Plasma Membrane Recycling Systems

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.     

Myosin 1G Is an Abundant Class I Myosin in Lymphocytes Whose Localization at the Plasma Membrane Depends on Its Ancient Divergent Pleckstrin Homology (PH) Domain (Myo1PH)

Class I myosins, which link F-actin to membrane, are largely undefined in lymphocytes. Mass spectrometric analysis of lymphocytes identified two short tail forms: (Myo1G and Myo1C) and one long tail (Myo1F). We investigated Myo1G, the most abundant in T-lymphocytes, and compared key findings with Myo1C and Myo1F. Myo1G localizes to the plasma membrane and associates in an ATP-releasable manner to the actin-containing insoluble pellet. The IQ+tail region of Myo1G (Myo1C and Myo1F) is sufficient for membrane localization, but membrane localization is augmented by the motor domain. The minimal region lacks IQ motifs but includes: 1) a PH-like domain; 2) a “Pre-PH” region; and 3) a “Post-PH” region. The Pre-PH predicted α helices may contribute electrostatically, because two conserved basic residues on one face are required for optimal membrane localization. Our sequence analysis characterizes the divergent PH domain family, Myo1PH, present also in long tail myosins, in eukaryotic proteins unrelated to myosins, and in a probable ancestral protein in prokaryotes. The Myo1G Myo1PH domain utilizes the classic lipid binding site for membrane association, because mutating either of two basic residues in the “signature motif” destroys membrane localization. Mutation of each basic residue of the Myo1G Myo1PH domain reveals another critical basic residue in the β3 strand, which is shared only by Myo1D. Myo1G differs from Myo1C in its phosphatidylinositol 4,5-bisphosphate dependence for membrane association, because membrane localization of phosphoinositide 5-phosphatase releases Myo1C from the membrane but not Myo1G. Thus Myo1PH domains likely play universal roles in myosin I membrane association, but different isoforms have diverged in their binding specificity.     

Human Exonuclease 5 Is a Novel Sliding Exonuclease Required for Genome Stability

Previously, we characterized Saccharomyces cerevisiae exonuclease 5 (EXO5), which is required for mitochondrial genome maintenance. Here, we identify the human homolog (C1orf176; EXO5) that functions in the repair of nuclear DNA damage. Human EXO5 (hEXO5) contains an iron-sulfur cluster. It is a single-stranded DNA (ssDNA)-specific bidirectional exonuclease with a strong preference for 5′-ends. After loading at an ssDNA end, hEXO5 slides extensively along the ssDNA prior to cutting, hence the designation sliding exonuclease. However, the single-stranded binding protein human replication protein A (hRPA) restricts sliding and enforces a unique, species-specific 5′-directionality onto hEXO5. This specificity is lost with a mutant form of hRPA (hRPA-t11) that fails to interact with hEXO5. hEXO5 localizes to nuclear repair foci in response to DNA damage, and its depletion in human cells leads to an increased sensitivity to DNA-damaging agents, in particular interstrand cross-linking-inducing agents. Depletion of hEXO5 also results in an increase in spontaneous and damage-induced chromosome abnormalities including the frequency of triradial chromosomes, suggesting an additional defect in the resolution of stalled DNA replication forks in hEXO5-depleted cells.     

Mitochondrial Enzyme Rhodanese Is Essential for 5 S Ribosomal RNA Import into Human Mitochondria

5 S rRNA is an essential component of ribosomes. In eukaryotic cells, it is distinguished by particularly complex intracellular traffic, including nuclear export and re-import. The finding that in mammalian cells 5 S rRNA can eventually escape its usual circuit toward nascent ribosomes to get imported into mitochondria has made the scheme more complex, and it has raised questions about both the mechanism of 5 S rRNA mitochondrial targeting and its function inside the organelle. Previously, we showed that import of 5 S rRNA into mitochondria requires unknown cytosolic proteins. Here, one of them was identified as mitochondrial thiosulfate sulfurtransferase, rhodanese. Rhodanese in its misfolded form was found to possess a strong and specific 5 S rRNA binding activity, exploiting sites found earlier to function as signals of 5 S rRNA mitochondrial localization. The interaction with 5 S rRNA occurs cotranslationally and results in formation of a stable complex in which rhodanese is preserved in a compact enzymatically inactive conformation. Human 5 S rRNA in a branched Mg²⁺-free form, upon its interaction with misfolded rhodanese, demonstrates characteristic functional traits of Hsp40 cochaperones implicated in mitochondrial precursor protein targeting, suggesting that it may use this mechanism to ensure its own mitochondrial localization. Finally, silencing of the rhodanese gene caused not only a proportional decrease of 5 S rRNA import but also a general inhibition of mitochondrial translation, indicating the functional importance of the imported 5 S rRNA inside the organelle.     

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S(1518)A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S(1518)E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25 degrees C but not at 36 degrees C. GFP-Myo2S(1518)E rings persisted at 36 degrees C in cdc7-24 but not in another SIN kinase mutant, sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy of myo2(+) was fused to GFP (strain myo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36 degrees C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.     

A Novel Plant Kinesin-Related Protein Specifically Associates with the Phragmoplast Organelles

In higher plants, the formation of the cell plate during cytokinesis requires coordinated microtubule (MT) reorganization and vesicle transport in the phragmoplast. MT-based kinesin motors are important players in both processes. To understand the mechanisms underlying plant cytokinesis, we have identified AtPAKRP2 (for Arabidopsis thaliana phragmoplast-associated kinesin-related protein 2). AtPAKRP2 is an ungrouped N-terminal motor kinesin. It first appeared in a punctate pattern among interzonal MTs during late anaphase. When the phragmoplast MT array appeared in a mirror pair, AtPAKRP2 became more concentrated near the division site, and additional signal could be detected elsewhere in the phragmoplast. In contrast, the previously identified AtPAKRP1 protein is associated specifically with bundles of MTs in the phragmoplast at or near their plus ends. Localization of the tobacco homolog(s) of AtPAKRP2 was altered by treatment of brefeldin A in BY-2 cells. We discuss the possibility that AtPAKRP1 plays a role in establishing and/or maintaining the phragmoplast MT array, and AtPAKRP2 may contribute to the transport of Golgi-derived vesicles in the phragmoplast.