Chapter 19 Fluorescence Microscopy Methods for Yeast

Pringle, John R.; Preston, Robert A.; Adams, A.E.; Stearns, Tim; Drubin, David G.; Haarer, Brian K.; Jones, Elizabeth W.

doi:10.1016/s0091-679x(08)61620-9

Cited by 531 publications

(481 citation statements)

References 199 publications

Supporting

Mentioning

468

Contrasting

Unclassified

Order By: Relevance

“…Affinity purification, immunoblotting, and immunofluorescence were done according to conventional procedures (Harlow and Lane, 1988;Pringle et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Chromosome Condensation Factor Brn1p Is Required for Chromatid Separation in Mitosis

Ouspenski

Cabello

Brinkley

2000

MBoC

100

View full text Add to dashboard Cite

This work describes BRN1, the budding yeast homologue of Drosophila Barren and Xenopus condensin subunit XCAP-H. The Drosophila protein is required for proper chromosome segregation in mitosis, and Xenopus protein functions in mitotic chromosome condensation. Mutant brn1 cells show a defect in mitotic chromosome condensation and sister chromatid separation and segregation in anaphase. Chromatid cohesion before anaphase is properly maintained in the mutants. Some brn1 mutant cells apparently arrest in S-phase, pointing to a possible function for Brn1p at this stage of the cell cycle. Brn1p is a nuclear protein with a nonuniform distribution pattern, and its level is up-regulated at mitosis. Temperature-sensitive mutations of BRN1 can be suppressed by overexpression of a novel gene YCG1, which is homologous to another Xenopus condensin subunit, XCAP-G. Overexpression of SMC2, a gene necessary for chromosome condensation, and a homologue of the XCAP-E condensin, does not suppress brn1, pointing to functional specialization of components of the condensin complex.

show abstract

“…Affinity purification, immunoblotting, and immunofluorescence were done according to conventional procedures (Harlow and Lane, 1988;Pringle et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

Chromosome Condensation Factor Brn1p Is Required for Chromatid Separation in Mitosis

Ouspenski

Cabello

Brinkley

2000

MBoC

100

View full text Add to dashboard Cite

show abstract

“…The immunofluorescence experiments were performed essentially as described by Pringle et al (1989). Cells were grown to exponential phase (OD 600 ϭ 0.5-0.8, 3-4 ϫ 10 7 /ml) and fixed directly for 4 h with formaldehyde (final concentration 5%).…”

Section: Immunofluorescence and Gfp Stainingmentioning

confidence: 99%

Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

Losko

Kopp

Kranz

et al. 2001

MBoC

View full text Add to dashboard Cite

Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitinationdependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.

show abstract

“…Yeast cultures were grown in SD medium to OD600 of 0.1-0.2 and processed for immunofluorescence as described (Pringle et al, 1989(Pringle et al, , 1991. Srv2p localization was achieved using affinity-purified rabbit polyclonal antiserum JF155 raised against full-length Srv2p (gift from Dr. J.…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

Lila

Drubin

1997

MBoC

164

219

View full text Add to dashboard Cite

show abstract

Chapter 19 Fluorescence Microscopy Methods for Yeast

Cited by 531 publications

References 199 publications

Chromosome Condensation Factor Brn1p Is Required for Chromatid Separation in Mitosis

Chromosome Condensation Factor Brn1p Is Required for Chromatid Separation in Mitosis

Uptake of the ATP-Binding Cassette (ABC) Transporter Ste6 into the Yeast Vacuole Is Blocked in the doa4 Mutant

Evidence for physical and functional interactions among two Saccharomyces cerevisiae SH3 domain proteins, an adenylyl cyclase-associated protein and the actin cytoskeleton.

Contact Info

Product

Resources

About