[40] Immunofluorescence methods for yeast

Pringle, John R.; Adams, A.E.; Drubin, David G.; Haarer, Brian K.

doi:10.1016/0076-6879(91)94043-c

Cited by 696 publications

(519 citation statements)

References 111 publications

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“…The GST-Ycf1p fusion protein was purified by standard techniques and used to immunize rabbits. The antiserum was affinity purified as described previously (Pringle et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Wemmie

Moye‐Rowley

1997

Molecular Microbiology

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SummaryYcf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15, vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein.These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP.

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“…The GST-Ycf1p fusion protein was purified by standard techniques and used to immunize rabbits. The antiserum was affinity purified as described previously (Pringle et al, 1991).…”

Section: Methodsmentioning

confidence: 99%

Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Wemmie

Moye‐Rowley

1997

Molecular Microbiology

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“…After washing three times with 5 mL each of PBST, the sample was treated with a 20±50 mL aliquot of 500-fold diluted second-step goat anti-rabbit IgG antibodies conjugated with a¯uorescence compound Cy3 at room temperature for 1 h in the moisturecontrolled chamber. After washing three times with PBST, the immuno-strained sample was stained with 10 mL of DAPI (4 H ,6-diamidino-2-phenylindole dihydrochloride, 10 mg/mL) for 1 min, and covered with one drop of mounting medium (1 mg/mL p-phenylenediamine, 90% glycerol in PBS [pH 9.0]) (Pringle et al 1991). The immuno-stained samples were observed with a¯uorescence and phase-contrast microscope (100ó bjective lens; Nikon, Japan) equipped with a chilled, colour 3CCD camera, C5810-01 (Hamamatsu, Japan), connected to a computer.…”

Section: Indirect Immuno¯uorescence Microscopymentioning

confidence: 99%

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

2000

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Background: The genome DNA of Escherichia coli is folded into the nucleosome-like structure, often called a nucleoid, by the binding of several DNAbinding proteins. We previously determined the speci®city and af®nity of DNA-binding for 12 species of the E. coli DNA-binding protein, and their intracellular concentrations at various growth phases. The intracellular localization of these proteins in E. coli could be predicted from these data, but no attempt has been made thus far to directly observe the intracellular distribution of the DNA-binding proteins.

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“…Overall cell morphologies were observed by differential interference contrast (DIC) microscopy, and indirect immunofluorescence was performed as described by Pringle et al (1991). Cdc11p was visualized using rabbit polyclonal antibodies (Ford and Pringle, 1991).…”

Section: Microscopymentioning

confidence: 99%

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

Caviston

Longtine

Pringle

et al. 2003

MBoC

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228

281

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The septins are a conserved family of GTP-binding, filament-forming proteins. In the yeast Saccharomyces cerevisiae, the septins form a ring at the mother-bud neck that appears to function primarily by serving as a scaffold for the recruitment of other proteins to the neck, where they participate in cytokinesis and a variety of other processes. Formation of the septin ring depends on the Rho-type GTPase Cdc42p but appears to be independent of the actin cytoskeleton. In this study, we investigated further the mechanisms of septin-ring formation. Fluorescence-recovery-after-photobleaching (FRAP) experiments indicated that the initial septin structure at the presumptive bud site is labile (exchanges subunits freely) but that it is converted into a stable ring as the bud emerges. Mutants carrying the cdc42 V36G allele or lacking two or all three of the known Cdc42p GTPase-activating proteins (GAPs: Bem3p, Rga1p, and Rga2p) could recruit the septins to the cell cortex but were blocked or delayed in forming a normal septin ring and had accompanying morphogenetic defects. These phenotypes were dramatically enhanced in mutants that were also defective in Cla4p or Gin4p, two protein kinases previously shown to be important for normal septin-ring formation. The Cdc42p GAPs colocalized with the septins both early and late in the cell cycle, and overexpression of the GAPs could suppress the septin-organization and morphogenetic defects of temperature-sensitive septin mutants. Taken together, the data suggest that formation of the mature septin ring is a process that consists of at least two distinguishable steps, recruitment of the septin proteins to the presumptive bud site and their assembly into the stable septin ring. Both steps appear to depend on Cdc42p, whereas the Cdc42p GAPs and the other proteins known to promote normal septin-ring formation appear to function in a partially redundant manner in the assembly step. In addition, because the eventual formation of a normal septin ring in a cdc42 V36G or GAP mutant was invariably accompanied by a switch from an abnormally elongated to a more normal bud morphology distal to the ring, it appears that the septin ring plays a direct role in determining the pattern of bud growth. INTRODUCTIONThe septin family of proteins was first recognized in yeast and now appears to be ubiquitous in fungi and animals, although not in plants (Longtine et al., 1996;Field and Kellogg, 1999;Trimble, 1999;Nguyen et al., 2000;Momany et al., 2001;Macara et al., 2002). Typical septins have a variable N-terminal region, a conserved core that includes the elements of a GTP-binding site, and a variable C-terminal region that (in all but a few cases) includes a predicted coiled-coil domain. In each species examined to date, there are two or more septins that form heterooligomeric complexes to perform their specific functions. Purified septins from yeast, Drosophila, Xenopus, and mammalian cells form filaments of 7-9 nm diameter and of variable length (Field et al., 1996;Frazier et al., 1998;Hsu et al...

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[40] Immunofluorescence methods for yeast

Cited by 696 publications

References 111 publications

Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Mutational analysis of the Saccharomyces cerevisiae ATP‐binding cassette transporter protein Ycf1p

Two types of localization of the DNA‐binding proteins within the Escherichia coli nucleoid

The Role of Cdc42p GTPase-activating Proteins in Assembly of the Septin Ring in Yeast

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