Mechanisms for targeting of the Saccharomyces cerevisiae GPI-anchored cell wall protein Crh2p to polarised growth sites
Jose M. Rodriguez-Peña1,
Cristina Rodriguez1,
Alberto Alvarez2,
César Nombela1 and
Javier Arroyo1,*
1 Departamento de Microbiología II, Facultad de Farmacia, Universidad
Complutense de Madrid, 28040 Madrid, Spain
2 Centro de Citometría de Flujo y Microscopía Confocal,
Universidad Complutense de Madrid, 28040 Madrid, Spain
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Fig. 1. Localisation of Crh2p-GFP in wild-type and bud1 cells. Crh2p-GFP
was examined by confocal microscopy in exponentially growing wild-type cells
transformed with the pJV40U plasmid (FY1679, see
Table 1) (A) and bud1
(JC223) cells bearing the plasmid pJV40L (B).
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Fig. 2. Crh2p-GFP distribution in a cdc42-1ts strain.
cdc42-1ts cells containing the pJV40G construct were
incubated either at permissive growth conditions (24°C) (A) or under
restrictive growth conditions (37°C for 5 hours) (B). Polarised
distribution of Crh2p was lacking at the restrictive temperature.
cdc42-1ts cells transformed with the pJV40G (Crh2-GFP) and
YCp(CDC42Sc) showed a normal polarised localisation pattern at the
restrictive temperature (C). Lysed cells were stained with propidium
iodide.
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Fig. 3. Localisation of Crh2p-GFP at the mother-bud neck depends on septin
integrity. Crh2p-GFP was followed by confocal microscopy in cdc10-11
cells (VCY1) growing at permissive conditions (24°C) (A). White arrowheads
indicate the typical ring-like distribution of the protein in small and medium
budden cells. Cellular localisation of Crh2p-GFP in the cdc10-11
(C-E) and the isogenic wild-type cells (strain 1784) (B) growing at the
restrictive temperature (37°C). Yellow arrowheads indicate the signal of
fluorescence detected in the enlarged mother-bud neck region in different
stages of the bud development. Two different slides of the same cells are
shown in D.
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Fig. 4. Localisation of Crh2p-GFP in a cdc15-lyt1 strain. Crh2p-GFP was
examined by confocal microscopy in cdc15-lyt1 cells (L2C24d strain)
growing at the permissive temperature (24°C) (A) or 5 hours after shifting
to the restrictive temperature (37°C) (B). Yellow arrowheads indicate the
presence of Crh2p-GFP in a conspicuous ring at the mother-bud neck of cells
expressing the mutant phenotype. In those cells that do not express the mutant
phenotype the fusion protein is correctly localised at the septum region (B,
bottom-left).
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Fig. 5. Bni4p is required for the proper Crh2p localisation during late stages of
the cell cycle. bni4 cells (10510A strain) were transformed with the
plasmid pJV40U and grown at 28°C. Crh2p-GFP localise to the site of bud
emergence during early stages of the cell cycle (A), but no Crh2p was present
at the septum during cytokinesis (B), as indicated by yellow arrows.
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Fig. 6. Localisation of Crh2p-GFP and Cwp1-GFP in a chs5 strain. Wild-type
cells (JM95 strain) transformed with the plasmid pJV40U (Crh2p-GFP) (A) and
chs5 cells (JM96 strain) transformed with the same plasmid (B-C) or
the plasmid pAR214 (Cwp1-GFP) (D) were grown at 28°C and analysed by
confocal microscopy.
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Fig. 7. Crh2p-GFP localisation depends on the presence of Sbe2p and Sbe22p.
Crh2-GFP was followed in wild-type (Y603 strain) (A) and sbe2 sbe22
cells (Y1949 strain) (B) growing at 24°C. Crh2p was properly distributed
in wild-type cells marking septa and bud scars. However the fusion protein
completely mislocalised in sbe2 sbe22 cells. C shows the localisation
of Cwp1-GFP (plasmid pAR214) in sbe2 sbe22 cells growing at
24°C.
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© The Company of Biologists Ltd 2002
