An interaction between Sla1p and Sla2p plays a role in regulating actin dynamics and endocytosis in budding yeast

Gourlay, Campbell W.; Dewar, Hilary; Warren, Derek; Costa, Rosaria; Satish, Nilima; Ayscough, Kathryn R.

doi:10.1242/jcs.00454

Cited by 73 publications

(71 citation statements)

References 37 publications

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“…6A), enlarged and fragmented vacuoles are evident in ϳ30% of the doa4-10 cells grown at 36°C. As reported for sla1⌬ cells (44,46,59), thick cell walls were also evident in sla1-10 mother cells but not in the bud. Significant cell lysis was apparent, with discontinuities in cell wall structure visible at the mother-bud juncture (Fig.…”

Section: Doa4p Sla1p and Sla2psupporting

confidence: 77%

“…Sla1p is a component of cortical actin patches and plays a role in endocytosis (45,59). The interaction of Sla1p with Sla2p also affects endocytosis by regulating actin dynamics (46). The sla1-10 allele (Fig.…”

Section: Doa4p Sla1p and Sla2pmentioning

confidence: 99%

“…Doa4p also functions in the endocytic pathway to remove ubiquitin from plasma membrane proteins targeted for vacuolar degradation (43). Sla1p and Sla2p function in the assembly of cortical actin patches and the actin cytoskeleton (44,45) and in endocytosis (46).…”

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confidence: 99%

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The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

Fiorani

Reid

Schepis

et al. 2004

Journal of Biological Chemistry

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DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36°C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4⌬ cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.DNA topoisomerases catalyze changes in the linkage of DNA strands, allowing for DNA unwinding or decatenation during DNA replication, transcription, recombination, and chromosome segregation (1, 2). Eukaryotic DNA topoisomerase I (Top1p) transiently cleaves a single strand of duplex DNA and forms a covalent tyrosyl linkage with a 3Ј-phosphoryl DNA end. The covalent Top1p-DNA intermediate allows for DNA rotation while conserving the energy of the cleaved phosphodiester bond.DNA topoisomerase I is the cellular target of the antitumor drug, camptothecin (CPT), 1 which reversibly stabilizes the covalent Top1p-DNA complex by inhibiting DNA religation (3-5). The ternary CPT-Top1p-DNA complexes per se are insufficient to induce a cytotoxic response. Rather, collisions with advancing replication forks convert the drug-stabilized complexes into the irreversible DNA lesions that trigger cell cycle arrest and cell death. This model is consistent with the S-phase specificity of CPT and the observation that inhibition of DNA synthesis by aphidicolin abrogates the cytotoxic action of the drug (6).In the yeast Saccharomyces cerevisiae, DNA topoisomerase

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Section: Doa4p Sla1p and Sla2psupporting

confidence: 77%

Section: Doa4p Sla1p and Sla2pmentioning

confidence: 99%

See 1 more Smart Citation

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

Fiorani

Reid

Schepis

et al. 2004

Journal of Biological Chemistry

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“…Further details of this interaction have not been reported. In addition, Sla1 had been demonstrated to bind to Ysc84 with the interaction mediated by the SH3 domain of Ysc84 to a specific site PAMPAR in Sla1 (amino acids 195-200) (Dewar et al, 2002;Gourlay et al, 2003). These data suggest that a possible site responsible for inhibition would be the SH3 domain.…”

Section: Ysc84 Sh3 Domain Interacts With a Specific Consensus Site Inmentioning

confidence: 96%

The WASP Homologue Las17 Activates the Novel Actin-regulatory Activity of Ysc84 to Promote Endocytosis in Yeast

Robertson¹,

Allwood²,

Smith³

et al. 2009

MBoC

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Actin plays an essential role in many eukaryotic cellular processes, including motility, generation of polarity, and membrane trafficking. Actin function in these roles is regulated by association with proteins that affect its polymerization state, dynamics, and organization. Numerous proteins have been shown to localize with cortical patches of yeast actin during endocytosis, but the role of many of these proteins remains poorly understood. Here, we reveal that the yeast protein Ysc84 represents a new class of actin-binding proteins, conserved from yeast to humans. It contains a novel N-terminal actin-binding domain termed Ysc84 actin binding (YAB), which can bind and bundle actin filaments. Intriguingly, full-length Ysc84 alone does not bind to actin, but binding can be activated by a specific motif within the polyproline region of the yeast WASP homologue Las17. We also identify a new monomeric actin-binding site on Las17. Together, the polyproline region of Las17 and Ysc84 can promote actin polymerization. Using live cell imaging, kinetics of assembly and disassembly of proteins at the endocytic site were analyzed and reveal that loss of Ysc84 and its homologue Lsb3 decrease inward movement of vesicles consistent with a role in actin polymerization during endocytosis.

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“…Unroofed cortices were labeled with anti-actin-immunogold complexes for electron microscopy. sla1 mutant cells had large F-actin aggregates by fluorescence microscopy (Figure 6A, inset; Holtzman et al, 1993;Gourlay et al, 2003), and the cytoplasmic surfaces of the unroofed cells had large (ϳ500 -1000 nm) flattened arrays of actin filaments on the plasma membrane (Figure 6A). sla2 mutant cells also had large F-actin aggregates by fluorescence microscopy (Figure 6B, inset;Holtzman et al, 1993).…”

Section: Actin Ultrastructures In Mutant Cellsmentioning

confidence: 99%

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

Rodal

Kozubowski

Goode

et al. 2005

MBoC

174

162

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Budding yeast has been a powerful model organism for studies of the roles of actin in endocytosis and septins in cell division and in signaling. However, the depth of mechanistic understanding that can be obtained from such studies has been severely hindered by a lack of ultrastructural information about how actin and septins are organized at the cell cortex. To address this problem, we developed rapid-freeze and deep-etch techniques to image the yeast cell cortex in spheroplasted cells at high resolution. The cortical actin cytoskeleton assembles into conical or mound-like structures composed of short, cross-linked filaments. The Arp2/3 complex localizes near the apex of these structures, suggesting that actin patch assembly may be initiated from the apex. Mutants in cortical actin patch components with defined defects in endocytosis disrupted different stages of cortical actin patch assembly. Based on these results, we propose a model for actin function during endocytosis. In addition to actin structures, we found that septin-containing filaments assemble into two kinds of higher order structures at the cell cortex: rings and ordered gauzes. These images provide the first high-resolution views of septin organization in cells. INTRODUCTIONBudding yeast has been used for over 20 yr as a model organism to study cytoskeletal function because the genes encoding many components of the cytoskeleton are conserved and because yeast cells are readily amenable to parallel genetic, biochemical, and cell biological analyses. Despite rapid advances in defining the components of the yeast cytoskeleton, its ultrastructure has been difficult to image at the resolution of the electron microscope, with the exception of microtubules (O'Toole et al., 1999). This may be due to the low abundance of the cytoskeleton in yeast relative to mammalian cells, to the presence of a cell wall that complicates extraction procedures, and to high ribosome density in the yeast cytoplasm. As a result, there has been a conspicuous deficit in our understanding of the higher order organization of actin and septin polymers in vivo (Pruyne and Bretscher, 2000). Thus, although it is possible to rapidly identify and mutate cytoskeletal proteins in yeast, there is no method by which to examine the ultrastructural consequences of these mutations. This gap could be filled by developing an approach to image the yeast cytoskeleton at high resolution.The yeast actin cytoskeleton is organized into three distinct structures: motile cortical patches that are polarized in the growing bud, actin cables that run along the motherdaughter cell axis, and a contractile cytokinetic ring at the neck of dividing cells (reviewed in Pruyne and Bretscher, 2000). Cortical actin patches contain many of the actin-associated proteins common to all eukaryotes (Goode and Rodal, 2001) and are composed of actin filaments that undergo rapid turnover (Ayscough et al., 1997). Actin is essential for yeast endocytosis (Kubler and Riezman, 1993). A number of recent studies suggest that act...

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An interaction between Sla1p and Sla2p plays a role in regulating actin dynamics and endocytosis in budding yeast

Cited by 73 publications

References 37 publications

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

The WASP Homologue Las17 Activates the Novel Actin-regulatory Activity of Ysc84 to Promote Endocytosis in Yeast

Actin and Septin Ultrastructures at the Budding Yeast Cell Cortex

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