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First published online 22 May 2007
doi: 10.1242/jcs.005124

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Chitin synthase III requires Chs4p-dependent translocation of Chs3p into the plasma membrane

Instituto de Microbiología Bioquímica and Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca, 37007 Salamanca, Spain

View larger version (37K): [in this window] [in a new window]   Fig. 1. Intracellular distribution of Chs4p and Chs3p. (A,B) Chs4p-GFP and Chs3p-GFP were localized by fluorescence (A) and confocal microscopy (B) in CRM233 and CRM103 strains, respectively. (C) Subcellular fractionation in continuous sucrose gradients of cell extracts obtained from the strain JAY29 containing plasmid pRS314-CHS4-GFP. Protein amounts were determined in even fractions by western blot using specific antibodies. The graph shows the quantitative analysis of these western blots data.

View larger version (138K): [in this window] [in a new window]   Fig. 2. Intracellular distribution of Chs4p-GFP. (A) Localization of Chs4p-GFP in the indicated strains. Note the accumulation at the septum but also the defined staining of the cell periphery in mother and daughter cells. (B) Intracellular accumulation of Chs4p-GFP in the indicated strains after transfer of the cells to galactose. Arrowheads indicate Chs4p deposition along bud membrane. Numbers indicate the time (in minutes) after induction. Wt and chs3 were transformed with the pRS314-GAL1-CHS4-GFP plasmid. All the strains used in the figure are W303 derivatives.

View larger version (40K): [in this window] [in a new window]   Fig. 3. Localization of Chs4p590-GFP. (A,B) Chs4p-GFP and Chs4p590-GFP were expressed in the indicated strains, all of them W303 derivatives. Cells of the wild type (A) are shown at higher magnification to show details. Note the absence of plasma membrane staining in all Chs4590-GFP-containing cells. Arrowhead indicates neck localization in the double mutant. Note the high levels of fluorescence in the vacuole. Numbers in each picture indicate the percentage (n>300) of cells showing defined staining at the neck or at the bud. (C) Subcellular fractionations comparing Chs4p and Chs4p590 association to particulate (Po) and soluble (So) fractions with and without 4% urea treatment. The figure represents a western blot analysis using the 3xHA tagged version of each protein. Numbers below bands indicate the percentage of the band in each fraction after quantitative densitometry.

View larger version (58K): [in this window] [in a new window]   Fig. 4. Intracellular distribution of Chs3p in the chs4 mutant. (A) Localization of Chs3p-GFP in the indicated strains. Arrowheads indicate Chs3p-GFP localization at the neck in the chs4 strain. Note the reduced intensity of the label compared with the wild type. (B) immunofluorescence localization of Chs3p-3xHA in wild-type, chs4 and chs5 strains (Y1306 background). Note the high definition of staining in the wild type in contrast to the diffuse and dotted pattern in the chs4. Chs3p was retained prominently in Golgi vesicles in the chs5. Data from wild type and chs4 are also expressed in graph format as percentages (n=230/240) of cells with the indicated distribution pattern, referred to the number of cells with defined staining. Discrete distribution refers to cells presenting staining sharply defined at the plasma membrane; Diffuse, polarized staining not sharply defined; Bud, cells showing diffuse staining at bud periphery.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Immunodetection of Chs3p-HA and markers Pma1p and Pep12p in discontinuous sucrose gradients using cell extracts from CRM872 (chs3) and CRM278 (chs3 chs4) strains transformed with the pHV7-C-HA. Western blots and their quantitative analyses are shown. Data are from a single representative experiment, but average values for several experiments are indicated in the text.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Plasma membrane levels of Chs3p and biological activity. (A) Quantification of Chs3p-3xHA levels in the plasma membrane fraction after discontinuous sucrose gradients and western analysis as in Fig. 2C. Protein extracts were obtained from the JAY29 (chs4) strain transformed with the indicated plasmids. Data represent the amount of Chs3p-3xHA localized in fractions 5-7 corresponding to the plasma membrane and are the average of three experiments. They are compared with resistance to 0.025 mg/ml Calcofluor and chitin levels (nmoles/100 mg cells), measured as indicated in the Materials and Methods. Calcofluor staining of these strains is shown in the lower images. (B) Distribution of Chs3p-3xHA in discontinuous sucrose gradients using extracts obtained from the chs4 and pRS424-CHS4 strains as in A. Lower panel represents specific CSIII activity (mU) in each fraction.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Effect of YPT32 overexpression on chitin synthesis. (A) Calcofluor staining of the indicated strains. Upper row shows the strains transformed with the pRS314 plasmid and the lower row those transformed with pRS314-GAL1-YPT32. Cells were grown in galactose and stained as indicated. (B) Chs3p-GFP localization after YPT32 overexpression. All the indicated strains contained the YpLac111-CHS3-GFP plasmid and the additional indicated plasmids. Note that such overexpression does not modified Chs3p-GFP accumulation in the neck in either wt or chs4 strains. Cells were grown in galactose and observed microscopically as described.

View larger version (53K): [in this window] [in a new window]   Fig. 8. Chitin synthesis and Chs3p distribution after endocytosis blockade. (A) Calcofluor staining and Chs3p-GFP distribution visualized microscopically as described in independent cultures. The indicated strains (BY4742 background) were transformed with the YpLac111-CHS3-GFP plasmid and thus contained two functional copies of Chs3p. Note the uniform distribution of Chs3p-GFP along the plasma membrane in the end4 mutants regardless of the presence of Chs4p. (B) Chs3p-GFP distribution after Latrunculin A treatment. CRM103 (chs3), CRM892 (chs3 chs4) and CRM1077 (chs3 chs6) strains transformed with the YpLac111-CHS3-GFP plasmid were grown to early log phase and treated with 50 µM latrunculin for one hour. (C) Same as in B but with treatment with 15 µM latrunculin for 15 minutes. Note that the fluorescence intensity is not uniform along the cell surface.

View larger version (69K): [in this window] [in a new window]   Fig. 9. Chs4p distribution in the absence of endocytosis. Calcofluor staining and Chs4p distribution in the end4 chs4 mutant transformed with pRS315 plasmids containing either the CHS4-GFP or CHS4590-GFP constructs. Chs4p-GFP distribution did not change as a result of the end4 mutation, independently of the C-terminal region of the protein (compare with Fig. 3A). Yeast cells were grown at 25°C.

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