High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells

Magdolen, Viktor; Drubin, David G.; Mages, Günter; Bandlow, Wolfhard

doi:10.1016/0014-5793(93)81733-g

Cited by 76 publications

(65 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our previous results [29, and unpublished data] indicate that the ratio of actin to total protein in profilin null cells is roughly equivalent to that of wild-type cells, suggesting that G-actin concentrations are also unaffected by loss of profilin. The observation that overproduction of profilin in yeast can suppress the lethality associated with actin overproduction [14] indicates that, under such conditions, profilin may be sequestering actin monomers, and that elimination of profilin might be expected to cause a significant increase in the cellular F-actin concentration. A possible explanation for these apparent discrepancies is that profilin's primary role may be to stimulate actin polymerization, but that it is also capable of sequestering actin monomers, particularly when it and/or actin are overproduced.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Profilin is required for the normal timing of actin polymerization in response to thermal stress

Yeh

Haarer

1996

FEBS Letters

View full text Add to dashboard Cite

We have used a fluorometric assay to determine the relative amounts of polymerized actin (F-actin) in wild-type and profilin mutant yeast cells. Our results indicate that profilin plays a role in maintaining normal F-actin levels in response to shifts to high temperature. Cells lacking prof'din display a greater drop in F-actin levels upon such temperature shifts, and are slower to recover to initial F-actin levels than are wild-type cells. Interestingly, shifts to cold temperatures result in rapid increases of F-actin levels in wild-type and profilin null cells. We have further determined that shifting to high-osmolarity growth conditions causes a relatively slow decrease in F-actin levels in wild-type cells, and a small but rapid increase in the F-actin levels in profilin null cells. Profilin null cells contain normal concentrations of F-actin while growing exponentially at room temperature, indicating that profilin is not essential for maintaining F-actin concentrations during steady-state growth. Our data suggest that actin is inherently unstable in vivo at high temperatures, and that profilin helps to maintain actin in its filamentous state at these temperatures, perhaps by stimulating actin polymerization in a proper temporal and spatial fashion. direct interaction between yeast profilin and adenylate cyclase (S. Palmieri and B. Haarer, unpublished data).Despite these many interactions attributed to profilins from various species, it is still unclear which roles are central to profilin function, and how such properties relate to the in vivo control of the actin cytoskeleton. Convincing arguments can be made for an in vivo role as an inhibitor [13][14][15], or stimulator [8,16] of actin polymerization. Indeed, it is entirely likely that profilin could be playing both roles, depending on species, cell type, or temporal regulation within an individual cell. To further address profilin's potential in vivo functions, we have utilized a fluorometric assay to measure the relative filamentous actin content in cells before and after changing growth conditions. Our findings are consistent with profilin's playing a significant role in the stimulation of actin polymerization under conditions that are perturbing to the yeast actin cytoskeleton. Materials and methods

show abstract

Section: Discussionmentioning

confidence: 99%

“…Convincing arguments can be made for an in vivo role as an inhibitor [13][14][15], or stimulator [8,16] of actin polymerization. Indeed, it is entirely likely that profilin could be playing both roles, depending on species, cell type, or temporal regulation within an individual cell.…”

mentioning

confidence: 99%

Profilin is required for the normal timing of actin polymerization in response to thermal stress

Yeh

Haarer

1996

FEBS Letters

View full text Add to dashboard Cite

show abstract

“…Cofilin's role in the capping assay seems to be profilin related, although it is not clear whether enhanced actin filament disassembly is caused by additive profilin/cofilin effects or synergistic effects caused by profilactin-specific barbed-end gating by cofilin. Approximations for the concentrations of actin and profilin in yeast are 60 and 20 M, respectively (Magdolen et al, 1993). Presuming that the Factin/G-actin ratio in yeast is at least 1:1, as has been stated for other organisms (Korn, 1982;Hug et al, 1995), then the G-actin/profilin ratio would be expected to be ϳ3:2.…”

Section: Is Aip1p a Capping Protein?mentioning

confidence: 93%

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

Clark

Teply

Haarer

et al. 2006

MBoC

View full text Add to dashboard Cite

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal ␤-propeller and a secondary actin binding site lies in a comparable location on its C-terminal ␤-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding-and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N-and C-terminal propeller domains.

show abstract

“…Actin overproduction is lethal in S. cerevisiae (37,48). Therefore, to avoid isolating the ACT1 gene again, we used a multicopy library to clone the authentic MDP2 gene.…”

Section: The Actin Cytoskeleton Is Aberrant In Mdp2-1 Mutant Cellsmentioning

confidence: 99%

Mutations Altering the Mitochondrial-Cytoplasmic Distribution of Mod5p Implicate the Actin Cytoskeleton and mRNA 3′ Ends and/or Protein Synthesis in Mitochondrial Delivery

Żołądek

Vaduva

Hunter

et al. 1995

Molecular and Cellular Biology

View full text Add to dashboard Cite

). A mutant allele of MOD5 encoding a protein (Mod5p-I,KR6) located predominantly in mitochondria was constructed. Mutants defective in delivering Mod5p-I,KR6 to mitochondria were sought by selecting cells with increased cytosolic activity of this protein. Twenty-five mutants defining four complementation groups, mdp1, mdp2, mdp3, and mdp4, were found. They are unable to respire at 34؇C or to grow on glucose medium at 38؇C. Cell fractionation studies showed that mdp1, mdp2, and mdp3 mutants have an altered mitochondrial-cytoplasmic distribution of Mod5p. mdp2 can be suppressed by ACT1, the actin-encoding gene. The actin cytoskeleton organization is also aberrant in mdp2 cells. MDP2 is the same as VRP1 (S. Eukaryotic cells have organelles separated physically from one another by lipid bilayers and the cytosol. Most proteins have a single destination such as the nucleus, mitochondria, peroxisomes, lysosomes, or the cell surface. However, we and others have discovered a class of genes that encode sorting isozymes, enzymes found in more than one cellular compartment (references 9, 10, 13, 18, 22, 34, 49, and 50; see also references cited in references 28 and 80).The Saccharomyces cerevisiae MOD5 and CCA1 genes that encode the tRNA processing enzymes N 6 -(⌬ 2 )-isopentenyl PP i : tRNA isopentenyltransferase (IPP transferase) and ATP (CTP):tRNA nucleotidyltransferase, respectively, are unique in that the sorting isozymes they encode are found in two organelles (mitochondria and nuclei) as well as the cytosol (10, 79, 80). MOD5 codes for two proteins that differ from each other by the presence or absence of an amino-terminal extension (28,69). Translation initiation at the first AUG of the open reading frame (ORF) produces Mod5p-I, which is located in mitochondria and the cytosol; translation initiation at the second AUG at codon 12 gives rise to Mod5p-II, located in the cytosol and nucleus (10).The mechanisms that locate proteins to single subcellular destinations have been intensively investigated. The importance of cis-acting sequences on the proteins is clearly established. Inroads are being made in identifying components on the organelles that serve as receptors and import channels. Cytosolic factors play critical roles (for an example, see review in reference 72), and recent observations suggest that the site of a protein's synthesis can also be an important factor in dictating its final destination (reviewed in reference 77). Despite this general outline of protein location, there is a very incomplete understanding of how the cell manages intracellular traffic. The natural distribution and function of sorting isozymes in multiple cellular compartments should provide a powerful tool to apply new genetic approaches to studies of protein delivery. This is because the distribution of sorting isozymes is balanced between multiple destinations, and the balance can be altered by cis-acting mutations as shown by the phenotypes caused by changing ATGs of MOD5 and CCA1 (10,28,80). The balance should also be altered by mu...

show abstract

High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells

Cited by 76 publications

References 39 publications

Profilin is required for the normal timing of actin polymerization in response to thermal stress

Profilin is required for the normal timing of actin polymerization in response to thermal stress

A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

Mutations Altering the Mitochondrial-Cytoplasmic Distribution of Mod5p Implicate the Actin Cytoskeleton and mRNA 3′ Ends and/or Protein Synthesis in Mitochondrial Delivery

Contact Info

Product

Resources

About