Chapter 12 Induction, Selection, and Experimental Uses of Temperature-Sensitive and Other Conditional Mutants of Yeast

Pringle, John R.

doi:10.1016/s0091-679x(08)60959-0

Cited by 34 publications

(20 citation statements)

References 141 publications

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“…These ts mutants were constructed by site-directed mutagenesis of the regions of the PEX3 gene coding for each of the five amino acid blocks conserved in the known Pex3 proteins. Because ts mutations are generally thought to be missense mutations that affect protein structure (Pringle, 1975), amino acid residues to be mutated were chosen on their ability to change local secondary structure when mutated and on their degree of conservation among the Pex3 proteins ( Figure 1C).…”

Section: During De Novo Peroxisome Biogenesis Matrix Protein Sequestmentioning

confidence: 99%

Peroxisome Biogenesis Occurs in an Unsynchronized Manner in Close Association with the Endoplasmic Reticulum in Temperature-sensitiveYarrowia lipolyticaPex3p Mutants

Bascom

Chan

Rachubinski

2003

MBoC

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Pex3p is a peroxisomal integral membrane protein required early in peroxisome biogenesis, and Pex3p-deficient cells lack identifiable peroxisomes. Two temperature-sensitive pex3 mutant strains of the yeast Yarrowia lipolytica were made to investigate the role of Pex3p in the early stages of peroxisome biogenesis. In glucose medium at 16°C, these mutants underwent de novo peroxisome biogenesis and exhibited early matrix protein sequestration into peroxisome-like structures found at the endoplasmic reticulum-rich periphery of cells or sometimes associated with nuclei. The de novo peroxisome biogenesis seemed unsynchronized, with peroxisomes occurring at different stages of development both within cells and between cells. Cells with peripheral nascent peroxisomes and cells with structures morphologically distinct from peroxisomes, such as semi/circular tubular structures that immunostained with antibodies to peroxisomal matrix proteins and to the endoplasmic reticulum-resident protein Kar2p, and that surrounded lipid droplets, were observed during up-regulation of peroxisome biogenesis in cells incubated in oleic acid medium at 16°C. These structures were not detected in wild-type or Pex3p-deficient cells. Their role in peroxisome biogenesis remains unclear. Targeting of peroxisomal matrix proteins to these structures suggests that Pex3p directly or indirectly sequesters components of the peroxisome biogenesis machinery. Such a role is consistent with Pex3p overexpression producing cells with fewer, larger, and clustered peroxisomes.

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Section: During De Novo Peroxisome Biogenesis Matrix Protein Sequestmentioning

confidence: 99%

Peroxisome Biogenesis Occurs in an Unsynchronized Manner in Close Association with the Endoplasmic Reticulum in Temperature-sensitiveYarrowia lipolyticaPex3p Mutants

Bascom

Chan

Rachubinski

2003

MBoC

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“…Total carbohydrate was determined by the anthrone reaction (40) and is expressed as glucose equivalents. 396 TIIE JOURNAL Or CELL BIOLOGY -VOLUME 89,1981 Other Methods Yeast Strains Used in this Study * All strains were maintained as deep-frozen stocks as described previously (29) . $ Strains 5011-D3 and 5011-D6 were constructed by L. Hartwell, University of Washington (personal communication), from mutant ts5011, an A364A derivative (21), using a procedure analogous to that used to construct A364A-D5 from A364A (16) .…”

Section: Quantitative Chitin Measurementsmentioning

confidence: 99%

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle.

Sloat¹,

Adams²,

Pringle³

1981

The Journal of Cell Biology

252

213

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Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i .e ., increase in cell mass and cell volume) at restrictive temperature (36°C) but were unable to form buds . Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36°C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanateconjugated concanavalin A suggested that mannan incorporation was also delocali-Zed in mutant cells grown at 36°C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded cells as well as in unbudded cells.Growth of the mutant strains at temperatures <36°C revealed allele-specific differences in behavior . Two strains produced buds of abnormal shape during growth at 33°C. Moreover, these same strains displayed abnormal localization of budding sites when grown at 24°C (the normal permissive temperature for the mutants) ; in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses.Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape .Cellular morphogenesis is the process by which cellular form and spatial organization are generated . During the cell division cycle of the yeast Saccharomyces cerevisiae, cellular morphogenesis includes the following sequential events : (a) selection of a nonrandom site at which budding will occur (1,14,23,25, 41,46) ; (b) formation of a ring of chitin (the "bud scar") in the largely nonchitinous cell wall at that site (24, 34) ; (c) localization of new cell wall growth to the region bounded by the chitin ring, resulting in the appearance and selective growth of a bud (10,11,22,27,28); (d) localization of new cell wall growth to the tip of the growing bud (l0, 11, 27, 43); (e) cytokinesis and the formation of septal cell wall (6, 43) . In addition, it seems that periods of uniform growth of the bud cell wall precede and follow the period of tip growth (11) ; presumably, the relative amounts of tip growth and of uniform growth are adjusted to yield the normal ellipsoidal shape of the daughter cell (10, 11) .

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“…Much of our understanding of the fundamental aspects of cell division has relied on the analysis of TS mutations in yeast (Pringle 1975). In Drosophila melanogaster, the use of a TS allele of Notch allowed its various spatial and temporal functions to be defined (Shellenbarger and Mohler 1978;Presente et al 2001;Costa et al 2005).…”

Section: Oss-of-function Phenotypes Provide Criticalmentioning

confidence: 99%

Temperature-Sensitive Mutations Made Easy: Generating Conditional Mutations by Using Temperature-Sensitive Inteins That Function Within Different Temperature Ranges

Tan¹,

Chen²,

Foote

et al. 2009

Genetics

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Reversible and easy to use, temperature-sensitive (TS) mutations are powerful tools for studying gene function. However, TS alleles are rare and difficult to generate and identify, and this has limited their use in most multicellular organisms. We have generated and characterized 41 intein switches, temperature-sensitive Sce VMA mutations that splice only at the permissive temperatures to generate intact host proteins. At nonpermissive temperatures, they fail to splice, resulting in a loss of function of the proteins in which they reside. By inserting an intein switch into a protein of interest, one can turn on and off the activities of the engineered protein with a simple temperature shift. The 41 TS inteins function in five different temperature ranges, with permissive temperatures ranging from 18°to 30°. This collection makes it possible to choose a TS-intein switch according to the optimal growth temperature of an organism or to suit a special experimental design.

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Chapter 12 Induction, Selection, and Experimental Uses of Temperature-Sensitive and Other Conditional Mutants of Yeast

Cited by 34 publications

References 141 publications

Peroxisome Biogenesis Occurs in an Unsynchronized Manner in Close Association with the Endoplasmic Reticulum in Temperature-sensitiveYarrowia lipolyticaPex3p Mutants

Peroxisome Biogenesis Occurs in an Unsynchronized Manner in Close Association with the Endoplasmic Reticulum in Temperature-sensitiveYarrowia lipolyticaPex3p Mutants

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle.

Temperature-Sensitive Mutations Made Easy: Generating Conditional Mutations by Using Temperature-Sensitive Inteins That Function Within Different Temperature Ranges

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