scholarly journals A Type V Myosin (Myo2p) and a Rab-like G-Protein (Ypt11p) Are Required for Retention of Newly Inherited Mitochondria in Yeast Cells during Cell Division

2004 ◽  
Vol 15 (9) ◽  
pp. 3994-4002 ◽  
Author(s):  
Istvan R. Boldogh ◽  
Sharmilee L. Ramcharan ◽  
Hyeong-Cheol Yang ◽  
Liza A. Pon
Keyword(s):  
Yeast Cells ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Myosin V ◽  
Binding Partner ◽  
Mitochondrial Movement ◽  
Mitochondrial Motility ◽  
Mother Cells ◽  
Actin Cables ◽  
Type V

Two actin-dependent force generators contribute to mitochondrial inheritance: Arp2/3 complex and the myosin V Myo2p (together with its Rab-like binding partner Ypt11p). We found that deletion of YPT11, reduction of the length of the Myo2p lever arm (myo2-Δ6IQ), or deletion of MYO4 (the other yeast myosin V), had no effect on mitochondrial morphology, colocalization of mitochondria with actin cables, or the velocity of bud-directed mitochondrial movement. In contrast, retention of mitochondria in the bud was compromised in YPT11 and MYO2 mutants. Retention of mitochondria in the bud tip of wild-type cells results in a 60% decrease in mitochondrial movement in buds compared with mother cells. In ypt11Δ mutants, however, the level of mitochondrial motility in buds was similar to that observed in mother cells. Moreover, the myo2-66 mutant, which carries a temperature-sensitive mutation in the Myo2p motor domain, exhibited a 55% decrease in accumulation of mitochondria in the bud tip, and an increase in accumulation of mitochondria at the retention site in the mother cell after shift to restrictive temperatures. Finally, destabilization of actin cables and the resulting delocalization of Myo2p from the bud tip had no significant effect on the accumulation of mitochondria in the bud tip.

scholarly journals The Cooh-Terminal Domain of Myo2p, a Yeast Myosin V, Has a Direct Role in Secretory Vesicle Targeting

1999 ◽  
Vol 147 (4) ◽  
pp. 791-808 ◽  
Author(s):  
Daniel Schott ◽  
Jackson Ho ◽  
David Pruyne ◽  
Anthony Bretscher
Keyword(s):  
Secretory Vesicle ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Tail Domain ◽  
Myosin V ◽  
Direct Role ◽  
Rab Protein ◽  
Polarized Delivery ◽  
Actin Cables ◽  
Type V

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable–dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

scholarly journals Capping protein insulates Arp2/3-assembled actin patches from formins

2019 ◽  
Author(s):  
Ingrid Billault-Chaumartin ◽  
Sophie G. Martin
Keyword(s):  
Cell Wall ◽  
Sexual Reproduction ◽  
Yeast Cells ◽  
Self Organization ◽  
Myosin V ◽  
Capping Protein ◽  
Dual Identity ◽  
Fusion Defect ◽  
Low Levels ◽  
Actin Cables

AbstractHow actin structures of distinct identities and functions co-exist within the same environment is a critical self-organization question. Fission yeast cells have a simple actin cytoskeleton made of four structures: Arp2/3 assembles actin patches around endocytic pits; the formins For3, Cdc12 and Fus1 assemble actin cables, the cytokinetic ring during division, and the fusion focus during sexual reproduction, respectively. The focus concentrates the delivery of hydrolases by myosin V to digest the cell wall for cell fusion. We discovered that cells lacking capping protein (CP), a heterodimer that blocks barbed-end dynamics and associates with actin patches, exhibit a delay in fusion. Consistent with CP-formin competition for barbed-end binding, Fus1, F-actin and the linear filament marker tropomyosin hyper-accumulate at the fusion focus in absence of CP. However, myosin V and exocytic cargoes are diverted to ectopic foci and reduced at the fusion focus, which underlies the fusion defect. Remarkably, ectopic foci coincide with actin patches, which now contain low levels of Fus1. During mitotic growth, actin patches lacking CP similarly display a dual identity, as they accumulate the formins For3 and Cdc12 and are co-decorated by tropomyosin and the patch marker fimbrin. Thus, CP serves to protect Arp2/3-nucleated structures from formin activity.

Loss of the mitochondrial Hsp70 functions causes aggregation of mitochondria in yeast cells

2001 ◽  
Vol 114 (19) ◽  
pp. 3565-3574 ◽  
Author(s):  
Akemi Kawai ◽  
Shuh-ichi Nishikawa ◽  
Aiko Hirata ◽  
Toshiya Endo
Keyword(s):  
Protein Synthesis ◽  
Elevated Temperature ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Mitochondrial Matrix ◽  
Mitochondrial Hsp70

Ssc1p, a member of the Hsp70 family in the mitochondrial matrix of budding yeast, mediates protein import into mitochondria and prevents irreversible aggregation of proteins in the mitochondrial matrix during folding/assembly or at elevated temperature. Here, we show that functional inactivation of the mitochondrial Hsp70 system causes aggregation of mitochondria. When temperature-sensitive mitochondrial Hsp70 mutant cells were incubated at restrictive temperature, a tubular network of mitochondria was collapsed to form aggregates. Inhibition of protein synthesis in the cytosol did not suppress the mitochondrial aggregation and functional impairment of Tim23, a subunit of mitochondrial protein translocator in the inner membrane, did not cause mitochondrial aggregation. Therefore defects of the Hsp70 function in protein import into mitochondria or resulting accumulation of precursor forms of mitochondrial proteins outside the mitochondria are not the causal reason for the aberrant mitochondrial morphology. By contrast, deletion of Mdj1p, a functional partner for mitochondrial Hsp70 in prevention of irreversible protein aggregation in the matrix, but not in protein import into mitochondria, caused aggregation of mitochondria, which was enhanced at elevated temperature (37°C). The aggregation of mitochondria at 37°C was reversed when the temperature was lowered to 23°C unless protein synthesis was blocked. On the basis of these results, we propose that the mitochondrial matrix contains a protein that is responsible for the maintenance of mitochondrial morphology and requires mitochondrial Hsp70 for its function.

scholarly journals Organelle-cytoskeletal interactions: actin mutations inhibit meiosis-dependent mitochondrial rearrangement in the budding yeast Saccharomyces cerevisiae.

1995 ◽  
Vol 6 (10) ◽  
pp. 1381-1396 ◽  
Author(s):  
M G Smith ◽  
V R Simon ◽  
H O'Sullivan ◽  
L A Pon
Keyword(s):  
Maximum Velocity ◽  
Time Lapse ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Cell Periphery ◽  
Yeast Saccharomyces Cerevisiae ◽  
Thread Formation ◽  
Mitochondrial Motility ◽  
Mitochondrial Defects ◽  
Meiosis I

During early stages of meiosis I, yeast mitochondria fuse to form a single continuous thread. Thereafter, portions of the mitochondrial thread are equally distributed to daughter cells. Using time-lapse fluorescence microscopy and a membrane potential sensing dye, mitochondria are resolved as small particles at the cell periphery in pre-meiotic, living yeast. These organelles display low levels of movement. During meiosis I, we observed a threefold increase in mitochondrial motility. Mitochondrial movements were linear, occurred at a maximum velocity of 25 +/- 6.7 nm/s, and resulted in organelle collision and fusion to form elongated tubular structures. Mitochondria do not co-localize with microtubules. Destabilization of microtubules by nocodazole treatment has no significant effect on the rate and extent of thread formation. In contrast, yeast bearing temperature-sensitive mutations in the actin-encoding ACT1 gene (act1-3 and act1-133) exhibit abnormal mitochondrial aggregation, fragmentation, and enlargement as well as loss of mitochondrial motility. In act1-3 cells, mitochondrial defects and actin delocalization occur only at restrictive temperatures. The act1-133 mutation, which perturbs the myosin-binding site of actin without significantly affecting actin cytoskeletal structure in meiotic yeast, results in mitochondrial morphology and motility defects at restrictive and permissive temperatures. These studies support a role for the actin cytoskeleton in the control of mitochondrial position and movements in meiotic yeast.

Isolation and characterization of fission yeast mutants defective in the assembly and placement of the contractile actin ring

1996 ◽  
Vol 109 (1) ◽  
pp. 131-142 ◽  
Author(s):  
F. Chang ◽  
A. Woollard ◽  
P. Nurse
Keyword(s):  
Fission Yeast ◽  
Ring Formation ◽  
Cell Cycle Checkpoint ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Actin Ring ◽  
Division Site ◽  
Isolation And Characterization ◽  
Cycle Checkpoint ◽  
Actin Cables

Fission yeast cells divide by medial cleavage using an actin-based contractile ring. We have conducted a genetic screen for temperature-sensitive mutants defective in the assembly and placement of this actin ring. Six genes necessary for actin ring formation and one gene necessary for placement of the actin ring have now been identified. The genes can be further organized into different phenotypic groups, suggesting that the gene products may have different functions in actin ring formation. Mutants of cdc3 and cdc8, which encode profilin and tropomyosin respectively, display disorganized actin patches in all cells. cdc12 and cdc15 mutants display disorganized actin patches during mitosis, but normal interphase actin patterns. cdc4 and rng2 mutants display disorganized actin cables during mitosis, but normal interphase actin patterns. In mid1 mutants, the actin ring and septum are positioned at random locations and angles on the cell surface, although the nucleus is positioned normally, indicating that the mid1 gene product is required to couple the division site to the position of the nucleus. mid1 mutant cells may reveal a new cell cycle checkpoint in telophase that coordinates cell division and the proper distribution of nuclei. The actin ring forms medially in a beta-tubulin mutant, showing that actin ring formation and placement are not dependent on the mitotic spindle.

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

2009 ◽  
Vol 20 (12) ◽  
pp. 2909-2919 ◽  
Author(s):  
Jason M. Sheltzer ◽  
Mark D. Rose
Keyword(s):  
Plasma Membranes ◽  
Contact Region ◽  
Differential Sensitivity ◽  
Yeast Cells ◽  
Cell Contact ◽  
Temperature Sensitive ◽  
Type I ◽  
Actin Patch ◽  
Type V ◽  
Rho Gef

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.

scholarly journals Yeast mitochondria contain ATP-sensitive, reversible actin-binding activity.

1994 ◽  
Vol 5 (7) ◽  
pp. 807-818 ◽  
Author(s):  
D A Lazzarino ◽  
I Boldogh ◽  
M G Smith ◽  
J Rosand ◽  
L A Pon
Keyword(s):  
Outer Membrane Proteins ◽  
Subcellular Fractionation ◽  
Binding Activity ◽  
Yeast Cells ◽  
Actin Binding ◽  
Mitochondrial Outer Membrane ◽  
Temperature Sensitive ◽  
Yeast Mitochondria ◽  
Mother Cells

Sedimentation assays were used to demonstrate and characterize binding of isolated yeast mitochondria to phalloidin-stabilized yeast F-actin. These actin-mitochondrial interactions are ATP sensitive, saturable, reversible, and do not depend upon mitochondrial membrane potential. Protease digestion of mitochondrial outer membrane proteins or saturation of myosin-binding sites on F-actin with the S1 subfragment of skeletal myosin block binding. These observations indicate that a protein (or proteins) on the mitochondrial surface mediates ATP-sensitive, reversible binding of mitochondria to the lateral surface of microfilaments. Actin copurifies with mitochondria during subcellular fractionation and is released from the organelle upon treatment with ATP. Thus, actin-mitochondrial interactions resembling those observed in vitro may also exist in intact yeast cells. Finally, a yeast mutant bearing a temperature-sensitive mutation in the actin-encoding ACT1 gene (act1-3) displays temperature-dependent defects in transfer of mitochondria from mother cells to newly developed buds during yeast cell mitosis.

scholarly journals A role for unsaturated fatty acids in mitochondrial movement and inheritance.

1991 ◽  
Vol 115 (5) ◽  
pp. 1249-1257 ◽  
Author(s):  
L C Stewart ◽  
M P Yaffe
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Unsaturated Fatty Acids ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Prolonged Incubation ◽  
Mitochondrial Movement ◽  
Mitochondrial Transfer ◽  
Mitochondrial Distribution

Yeast cells with the mdm2 mutation display temperature-sensitive growth and defective intracellular mitochondrial movement at the non-permissive temperature. The latter phenotype includes both an absence of mitochondrial transfer into daughter buds of mitotically growing cells and an aberrant mitochondrial distribution in cells exposed to mating pheromone. The wild-type MDM2 gene was cloned by complementation, and DNA sequence analysis revealed a large open reading frame encoding a putative protein of 58.4 kD. The predicted protein sequence is identical to that reported for the yeast OLE1 gene encoding fatty acid desaturase. Unsaturated fatty acid levels are substantially decreased in mdm2 cells after a prolonged incubation at the non-permissive temperature. The addition of oleic acid complements the temperature-sensitive growth and mitochondrial distribution defects of the mutant cells. These results indicate that mdm2 is a temperature-sensitive allele of OLE1 and demonstrate an essential role for unsaturated fatty acids in mitochondrial movement and inheritance.

scholarly journals Suppressors of mdm20 in Yeast Identify New Alleles of ACT1 and TPM1 Predicted to Enhance Actin-Tropomyosin Interactions

Genetics ◽  
2000 ◽  
Vol 156 (2) ◽  
pp. 523-534
Author(s):  
Jason M Singer ◽  
Greg J Hermann ◽  
Janet M Shaw
Keyword(s):  
Yeast Cells ◽  
Temperature Sensitive ◽  
Mitochondrial Inheritance ◽  
Actin Organization ◽  
Growth Defects ◽  
Enhanced Sensitivity ◽  
Suppressor Mutations ◽  
Actin Cables ◽  
Actin Protein ◽  
New Alleles

Abstract The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.

scholarly journals A Protein Complex Containing Mdm10p, Mdm12p, and Mmm1p Links Mitochondrial Membranes and DNA to the Cytoskeleton-based Segregation Machinery

2003 ◽  
Vol 14 (11) ◽  
pp. 4618-4627 ◽  
Author(s):  
Istvan R. Boldogh ◽  
Dan W. Nowakowski ◽  
Hyeong-Cheol Yang ◽  
Haesung Chung ◽  
Sharon Karmon ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Reciprocal Relationship ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Mitochondrial Movement ◽  
Close Proximity ◽  
Mtdna Deletion ◽  
Mitochondrial Motility ◽  
In Cells

Previous studies indicate that two proteins, Mmm1p and Mdm10p, are required to link mitochondria to the actin cytoskeleton of yeast and for actin-based control of mitochondrial movement, inheritance and morphology. Both proteins are integral mitochondrial outer membrane proteins. Mmm1p localizes to punctate structures in close proximity to mitochondrial DNA (mtDNA) nucleoids. We found that Mmm1p and Mdm10p exist in a complex with Mdm12p, another integral mitochondrial outer membrane protein required for mitochondrial morphology and inheritance. This interpretation is based on observations that 1) Mdm10p and Mdm12p showed the same localization as Mmm1p; 2) Mdm12p, like Mdm10p and Mmm1p, was required for mitochondrial motility; and 3) all three proteins coimmunoprecipitated with each other. Moreover, Mdm10p localized to mitochondria in the absence of the other subunits. In contrast, deletion of MMM1 resulted in mislocalization of Mdm12p, and deletion of MDM12 caused mislocalization of Mmm1p. Finally, we observed a reciprocal relationship between the Mdm10p/Mdm12p/Mmm1p complex and mtDNA. Deletion of any one of the subunits resulted in loss of mtDNA or defects in mtDNA nucleoid maintenance. Conversely, deletion of mtDNA affected mitochondrial motility: mitochondria in cells without mtDNA move 2–3 times faster than mitochondria in cells with mtDNA. These observations support a model in which the Mdm10p/Mdm12p/Mmm1p complex links the minimum heritable unit of mitochondria (mtDNA and mitochondrial outer and inner membranes) to the cytoskeletal system that drives transfer of that unit from mother to daughter cells.

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