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. 1999 Jun 14;145(6):1153-63.
doi: 10.1083/jcb.145.6.1153.

Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae

J A Trilla  1 , A Durán, C Roncero
Affiliations

Chs7p, a new protein involved in the control of protein export from the endoplasmic reticulum that is specifically engaged in the regulation of chitin synthesis in Saccharomyces cerevisiae

J A Trilla et al. J Cell Biol. .

Erratum in

  • J Cell Biol 1999 Jul 12;146(1):following 264

Abstract

The Saccharomyces cerevisiae CHS7 gene encodes an integral membrane protein located in the ER which is directly involved in chitin synthesis through the regulation of chitin synthase III (CSIII) activity. In the absence of CHS7 product, Chs3p, but not other secreted proteins, is retained in the ER, leading to a severe defect in CSIII activity and consequently, to a reduced rate of chitin synthesis. In addition, chs7 null mutants show the yeast phenotypes associated with a lack of chitin: reduced mating efficiency and lack of the chitosan ascospore layer, clear indications of Chs7p function throughout the S. cerevisiae biological cycle. CHS3 overexpression does not lead to increased levels of CSIII because the Chs3p excess is retained in the ER. However, joint overexpression of CHS3 and CHS7 increases the export of Chs3p from the ER and this is accompanied by a concomitant increase in CSIII activity, indicating that the amount of Chs7p is a limiting factor for CSIII activity. Accordingly, CHS7 transcription is increased when elevated amounts of chitin synthesis are detected. These results show that Chs7p forms part of a new mechanism specifically involved in Chs3p export from the ER and consequently, in the regulation of CSIII activity.

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Figures

Figure 1
Figure 1
Identification and characteristics of Chs7p. (A) Subcloning strategy used in the identification of YHR142 as complementing gene for the cwr6 mutants. Complementation was determined by analyzing the reversal of Calcofluor resistance in cwr6 cells. Useful restriction sites are indicated: E, EcoRI; C, ClaI; V, EcoRV; X, XbaI; St, StuI; B, BamHI; P, PstI; K, KpnI; and S, SalI. (B) Amino acid sequence of Chs7p. Membrane-spanning domains are underlined. (C) Kyte-Doolittle hydrophobicity plot of Chs7p.
Figure 2
Figure 2
Transcription levels of CHS7. Northern blots were done with RNAs obtained from cells grown under different conditions and loaded in equivalent amounts. CHS7 mRNA was detected using the NdeI–NdeI fragment as a probe (Materials and Methods). In all cases, autoradiographic images and quantitative determinations by PhosphorImager (Materials and Methods) are shown. Relative amounts of CHS7 mRNA are referred to actin levels (not shown) and normalized with respect to the value obtained at the respective control points (left lane of each figure). (A) RNA was obtained from X2180-1A cells incubated for the indicated times on 5 μM α-factor (left) or from cells grown for 2 h in the absence (−) or presence (+) of 50 μg/ml Calcofluor. (B) RNA was obtained from YPA24 diploid strain incubated in sporulation medium for the indicated times.
Figure 3
Figure 3
Localization of Chs7p. (A) JAY25 cells transformed with pTM15 (pRS316::CHS7-GFP) were visualized directly in a Zeiss laser-confocal microscope under appropriate excitation conditions. Note the continuous staining along the nuclear periphery and the patches associated with cytoplasmic membrane. Cells shown on the lower part of the figure are in the process of nuclear division. (B) Indirect immunofluorescence localization of Sec63p-Myc in wild-type (15Daub) cells transformed with pRS315::SEC63-MYC. (C) Western blot experiments showing subcellular distribution of several proteins after equilibrium centrifugation in a sucrose gradient. Fractions were collected from the bottom (lower fractions, higher densities). Equal volumes of each fraction were loaded per lane (Materials and Methods).
Figure 4
Figure 4
Characterization of Chs3p in wild-type and Δchs7 mutants. (A) Expression levels of Chs3p-3XHA in wild-type and Δchs7 strains. (B) Mobility of Chs3p-3XHA from wild-type and Δchs7 before (−) and after (+) EndoH treatment. Chs3p* indicates mobility of the protein after N-glycosylation removal. Protein extracts (A and B) were prepared from HVY374 (wild-type) and JAY25 (Δchs7) strains transformed with plasmid pHV7-CHS3::3XHA and detected by Western blot (Materials and Methods). (C) Chs3p-3XHA detected by indirect immunofluorescence with HA.11 antibody. Early logarithmically growing cells were processed as described in Materials and Methods. Strains: a, Y1306 (CHS3::3XHA); b and c, JAY27 (CHS3::3XHA, chs7::HIS3). (a) Polarized Chs3p distribution in wild-type cells. (b and c) Typical ER distribution of Chs3p in Δchs7 mutant. Note the irregular distribution (dotted staining) observed after higher magnification in c. Bars, 2 μm.
Figure 5
Figure 5
Immunolocalization of Chs3p-3XHA in chs mutants. All cells are Y1306 (CHS3::3XHA) with different chs mutations. (A) JAY29, chs4::URA3. (B) JAY31, chs4::URA3 chs7::HIS3. (C) JAY30, chs5::ADE2. (D) JAY33, chs5::ADE2 chs7::HIS3. (E) JAY28, chs6::URA3. (F) JAY32, chs6::URA3 chs7::HIS3. (A, C, and E) Polarized distribution of Chs3p at the base of the emerging bud (Fig. 4 A) is lost in chs mutants. Each chs mutant shows the typical Chs3p localization as described. (B, D, and F) In Δchs7 mutants, Chs3p-3XHA shows ER localization.
Figure 6
Figure 6
Secretion phenotypes of Δchs7 mutants. (A) Production of α-factor measured by the size of the growth inhibition halo. (B) Activities of several enzymatic activities in wild-type and chs7 null mutants. Activities were determined in HVY374 (WT) and JAY25 (chs7::HIS2) isogenic strains and expressed as described in Materials and Methods.
Figure 7
Figure 7
CSIII activity in strains overexpressing CHS genes. CSIII activity was determined in cell extracts with (total activity) or without (basal activity) trypsin activation (Materials and Methods). Numbers indicate the ratio between total and basal activity. The results are the average of three independent experiments. Standard deviation bars are shown. On the abscissa, the names of overexpressed CHS genes are indicated in each case. Overexpression was achieved by transforming wild-type (15Daub) cells with multicopy plasmids: pRS423::CHS3, pRS425:: CHS4, or pRS426::CHS7 (Materials and Methods).
Figure 8
Figure 8
Immunolocalization of overexpressed Chs3p-3XHA. (A) Characteristic ER staining of strain Y604 transformed with pRS423::CHS3-3XHA after immunodetection of Chs3p-3XHA. (B) Chs3p-3XHA localization in Y604 cotransformed with pRS423::CHS3-3XHA and pRS426::CHS7. Note the dotted staining in most cells, some showing polarized staining (arrows). Some cells also show residual perinuclear staining.
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