Vol. 52 No. 1/2005 207–220 QUARTERLY

Dimethylallyl diphosphate, an isomer of isopentenyl diphosphate, is a common substrate of Mod5p, a tRNA modifying enzyme, and the farnesyl diphosphate synthase Erg20p, the key enzyme of the isoprenoid pathway. rsp5 mutants, defective in the Rsp5 ubiquitin-protein ligase, were isolated and characterized as altering the mitochondrial/cytosolic distribution of Mod5p. To understand better how competition for the substrate determines the regulation at the molecular level, we analyzed the effect of the rsp5-13 mutation on Erg20p expression. The level of Erg20p was three times lower in rsp5-13 compared to the wild type strain and this effect was dependent on active Mod5p. Northern blot analysis indicated a regulatory role of Rsp5p in ERG20 transcription. ERG20 expression was also impaired in pkc1Delta lacking a component of the cell wall integrity signaling pathway. Low expression of Erg20p in rsp5 cells was accompanied by low level of ergosterol, the main end product of the isoprenoid pathway. Additionally, rsp5 strains were resistant to nystatin, which binds to ergosterol present in the plasma membrane, and sensitive to calcofluor white, a drug destabilizing cell wall integrity by binding to chitin. Furthermore, the cell wall structure appeared abnormal in most rsp5-13 cells investigated by electron microscopy and chitin level in the cell wall was increased two-fold. These results indicate that Rsp5p affects the isoprenoid pathway which has important roles in ergosterol biosynthesis, protein glycosylation and transport and in this way may influence the composition of the plasma membrane and cell wall.

The isoprenoid pathway supplies the cell with isoprenoid lipids, which are used to produce dolichols, prenylated proteins, ubiquinon and heme.However, the most prevailing product of the pathway is the ergosterol in yeast and cholesterol in humans.Ergosterol is an important component of the plasma membrane and organellar membranes which affects their fluidity, permeability and other features.Cells lacking normal ergosterol biosynthesis show defects in endocytosis (Munn et al., 1999;Heese-Peck et al., 2002), vacuole fusion (Kato & Wickner, 2001), and mitochondrial respiration (Parks & Casey, 1995).The branch point enzyme of the isoprenoid pathway is an essential farnesyl diphosphate synthase (Erg20p) which catalyses the sequential condensation of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP) and further farnesyl diphosphate (FPP) (Song & Poulter, 1994).FPP, besides being a precursor for several isoprenoid compounds, also serves a regulatory role in the isoprenoid pathway (see review by Grabińska & Palamarczyk, 2002).DMAPP used by Erg20p is also a common substrate for Mod5p, a tRNA isopentenyltransferase (Dihanich et al., 1987).Both enzymes compete for the DMAPP pool as indicated by the finding that overexpression of the ERG20 gene causes a decrease in isopentenylation of tRNAs (Benko et al., 2000).This was confirmed further by the result showing that an increase in tRNA biosynthesis was accompanied by lower ergosterol production if active Mod5p was present (Kamińska et al., 2002a).
Sterol depletion in mammalian cells causes activation of the transcription factors known as sterol regulatory element (SRE) binding proteins (SREBPs) (reviewed in Edwards et al., 2000).When sterols are abundant the SREBPs are inactive and tethered to the endoplasmic reticulum membrane by their transmembrane domains.When sterol level drops, regulated proteolysis releases the transcrpitional activation domain of SREBPs allowing its nuclear transport.SREBPs activate transcription of genes involved in sterol and fatty acid synthesis.The human gene encoding FPP synthase contains SRE sequence (Sato et al., 1996).However, less is known about this regulatory mechanism in yeast.Many genes of the isoprenoid pathway are transcriptionaly regulated in response to erg mutations, inhibitors of isoprenoid pathway and anaerobiosis as determined by genome-wide expression profile analysis (Dimster-Denk et al., 1999;Bammert & Fostel, 2000;Kwast et al., 2002;Agarwal et al., 2003).On the other hand, ERG20 is constitutively expressed after inhibition of the downstream part of the ergosterol synthesis pathway by azoles (Bammert & Fostel, 2000;Agarwal et al., 2003) and in anaerobiosis (Kwast et al., 2002), and only 2-3-fold upregulated by inhibition by lovastatin of HMG-CoA reductase from the upstream part of the pathway (Dimster-Denk et al., 1999).An about two-fold increase of ERG20 expression was also observed in an RNA polymerase III regulatory mutant that has enhanced tRNA synthesis (Kamiñska et al., 2002a).Many ERG genes are activated by the Upc2p and Ecm22p transcription factors which bind yeast SRE (Vik & Rine, 2001) and repressed by Mot3p and Rox1p (Kwast et al., 2002;Henry et al., 2002).However, ERG20 was not found among those ERG genes and the mech-anism of transcriptional regulation of ERG20 remains unknown.
One of the enzymes of the isoprenoid pathway, HMG-CoA reductase (Hmg2p), is physiologically regulated by ubiquitination and degradation in proteasomes (Gardner et al., 2001).Ubiquitination is a posttranslational modification of proteins by the polypeptide ubiquitin, which is achieved by the action of a cascade of enzymatic reactions involving ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3) which has an important role in substrate selection.Polyubiquitination serves to direct short-lived and abnormal proteins for proteasomal degradation but mono-and multiubiquitination does not involve proteasomes and has regulatory roles in several processes (reviewed in Weissman, 2001;Hershko & Ciechanover, 1998;Hicke & Dunn, 2003;Muratani & Tansey, 2003).One of the yeast ubiquitin ligases is Rsp5p (Huibregtse et al., 1995).The best studied is Rsp5p involvement in ubiquitination and endocytosis of plasma membrane transporters and receptors (reviewed in Rotin & Haguenauer-Tsapis, 2000; see also Gajewska et al., 2001;Kamińska et al., 2002b;Stamenova et al., 2004) but it affects also other steps of vesicular traffic (Katzmann et al., 2004;Morvan et al., 2004), transcription (Beaudenon et al., 1999), nuclear export of mRNAs and tRNAs (Rodriguez et al., 2003;Neumann et al., 2003;and our unpublished observations), biosynthesis of unsaturated fatty acids (Hoppe et al., 2000;Shcherbik et al., 2003;2004), regulation of cellular pH (De la Fuente et al., 1997;Kamiñska et al., 2000) and other processes.Genetic links were found between RSP5 gene and cellular signaling pathways.RSP5 is a multicopy suppressor of the temperature-sensitive phenotype of the rlm1 slt1(ssd1) double mutant (Watanabe et al., 1995) which contains mutations in the RLM1 gene encoding a transcription factor regulated by the Pkc1p signaling pathway and in the SSD1 gene encoding a signaling protein involved in cell wall integrity independent of Pkc1p (Kaeberleine & Guarente, 2002).Moreover, the WSC2 gene encoding one of the plasma membrane sensors of the Pkc1p-dependent pathway was identified as multicopy suppressor of rsp5 mutant phenotypes (De la Fuente & Portillo, 2000).
In this work the rsp5-13 mutant with deficiency of ubiquitin ligase was studied to investigate a possible link between the ubiquitination pathway, the expression of ERG20 gene encoding the branch point enzyme of the isoprenoid pathway, and sterol biosynthesis.This connection was suggested by isolating rsp5 mutants as affecting subcellular distribution of Mod5p, an enzyme using an isoprenoid substrate (Żołądek et al., 1995; 1997).Seeking for the consequences of this link we also studied the effect of the rsp5-13 mutation on cell wall structure and function.We show here a correlation between the rsp5-13 mutation, lower expression of ERG20 and lower synthesis of ergosterol.As a consequence of mutated Rsp5p the cell wall structure, chitin content and sensitivity to calcofluor white, a chitin-binding drug, was also changed.

MATERIALS AND METHODS
Yeast strains, genetics methods and media.Saccharomyces cerevisiae strains used are listed in Table 1.Standard genetic manipulations were performed as described by Sherman (2002).Yeast were grown on rich media containing 2% glucose (YEPD), or on SC-ura and SC-ura-leu selective media containing 2% glucose which were supplemented with required amino acids.Strain pkc1D (Y23133-1D) was grown in media additionally supplemented with 1 M sorbitol.In growth assays YEPD with addition of nystatin (Sigma, 6 U/ml) or calcofluor white (Sigma, 1 mg/ml) was also used.Cells were grown overnight in YEPD at 30 o C to equal absorbance, then ten-fold serially diluted and spotted on plates.
Ttoal protein extracts and Western blot analysis.Protein extracts were prepared by disrupting cells by alkaline lysis (Kamińska et al., 2002a).Samples were analyzed by standard Western blot method using polyclonal rabbit anti-ferrochelatase antibodies (kind gift of J. Rytka, IBB PAS, Warszawa, Poland) or rabbit anti-Mdm1p antibodies (from M. Yaffe, University of California, U.S.A.) and secondary anti-rabbit horseradish peroxidase-conjugated antibody followed by chemiluminescence (ECL Plus, Amersham).The level of proteins was quantified using Image Quant V.1.1 software with local average background subtraction.
Preparation of total RNA and Northern analysis.Preparation of total RNA and Northern analysis were performed using specific probes for ERG20 and ACT1 as described (Kamińska et al., 2002a).The levels of RNA were quantified using Gene Tools 3.0 (Syngene).
Determination of ergosterol levels.Sterols were extracted from yeast cells with n-heptane after alkaline lysis and the amount of ergosterol was determined from the absorbance at 281.5 nm (Servouse & Karst, 1986).
Transmission electron microscopy.Cells grown on YEPD to logarithmic phase were fixed with 2% glutaraldehyde followed by treatment with 2% OsO 4 for 2 h.Thin sections were cut from Epon blocks and post-stained with 2% uranyl acetate followed by lead citrate (Reynolds, 1963).Micrographs were 210 J. Kamińska and others 2005 Strain Genotype Source or Reference

Derivative of Y23133 Euroscarf
Table 1.List of S. cerevisiae strains taken on a JEOL transmission electron microscope JEM1220.

Analysis of cell wall composition.
To determine chitin and glucan levels cells were grown overnight in SC media supplemented with required amino acids, collected and disrupted with glass beads.Cell walls were separated from cell extracts by 5 000 ´g centrifugation.For chitin measurements alkaline lysis of cell walls was performed in 6% KOH for 90 min at 80 o C to release cell wall proteins.After neutralization with acetic acid the cell walls were washed with phosphate-buffered saline and chitinase buffer pH 6.0, containing 18 mM citric acid and 60 mM dibasic sodium phosphate, and then treated with chitinase C (InterSpex Products) for 3 h at 37 o C. Chitin was measured as described (Bulik et al., 2003).For glucan determination cell walls were hydrolysed similarly as described for Trichoderma viride (Nemčovič & Farkaš, 2001) and glucose in the hydrolysate was measured by a published method (Wood & Bhat, 1988).
Zymolyase assay.Sensitivity to zymolyase was determined based on the method described by De Groot et al. (2001).Cells grown overnight in YEPD were resuspended in 10 mM Tris/HCl, pH 7.4, than incubated at 37 o C with zymolyase 20T (ICN) 50 mg/ml and A 600 was measured in 15 min intervals.

The rsp5-13 mutation affects expression of ERG20 gene
Erg20p, a farnesyl diphosphate synthase, competes for the substrate, dimethylallyl diphosphate, with Mod5p (Benko et al., 2000), a tRNA isopentenyltransferase which modifies cytosolic and mitochondrial tRNAs (Dihanich et al., 1987).On the other hand, distribution of the cellular pool of Mod5p is dependent on the activity of Rsp5p, a protein-ubiquitin ligase (Żołądek et al., 1995; 1997).These two findings persuaded us to assess if Rsp5p affects the ergosterol pathway.For these studies we used the rsp5-13 mutant with the G707D substitution in the catalytic domain of Rsp5p (Żołądek et al., 1997) which confers a temperature-sensitivity phenotype.
The level of Erg20 protein was determined in isogenic strains rsp5-13, rsp5-13 mod5-1 and wild type transformed with pJK1 centromeric plasmid bearing the ProtA-ERG20 gene fusion.Cellular extracts prepared from cells grown at 28 o C and shifted to the restrictive temperature of 37 o C for 2.5 h were analyzed by Western blot using anti-ferrochelatase antibody.This antibody recognized ProtA and endogenous ferrochelatase, which served as internal control for quantification of ProtA-Erg20p bands.In rsp5-13 the level of Erg20p was decreased three fold as compared to wild type cells, both grown at 28 o C and shifted to the restrictive temperature (Fig. 1).However, this difference was dependent on the pres- Yeast strains: wt (T8-1D/1, pJK1), rsp5-13 (TZ23/1, pJK1), mod5-1 (T8-1D, pJK1) and mod5-1 rsp5-13 (TZ23, pJK1) were grown in SC-ura-leu at permissive temperature followed by a shift to 37 ence of active Mod5p.rsp5-13 had no effect on Erg20p level in the background of mod5-1 mutation which was probably due to the lack of competition of mutant Mod5-1p with Erg20p for the substrate.Moreover, an about 40% decrease of Erg20p level in response to temperature elevation was consistently observed in all strains studied.
Since the Erg20p cellular content was decreased in the rsp5-13 mutant strain, we determined if the reason for this effect was the difference in ERG20 transcript level.Total RNA isolated from rsp5-13 and wild type strain transformed with pJK1, grown at 28 o C and shifted to 37 o C for 2 h, was analyzed by Northern blot using ERG20 specific probe.As shown in Fig. 2, the level of both genomic ERG20 transcript and plasmid ProtA-ERG20 transcript were decreased 3-4-fold in the rsp5-13 mutant.In cells shifted to the elevated temperature the decrease of ERG20 transcript level was observed irrespective of the presence of the rsp5-13 mutation.Therefore, changes in ProtA-ERG20 and ERG20 transcript levels in response to the rsp5-13 mutation or temperature elevation exactly paralleled the changes observed in Erg20p level.From these results we concluded that Rsp5p is involved in regulation of ERG20 expression and this regulation occurs at the level of ERG20 transcript synthesis or degradation.

Transcription factors involved in expression of ERG20
Since the control of ERG20 expression by Rsp5p possibly occurred at the transcriptional level, the question arose which transcription factor was involved in this regulation.ERG20 promoter had not been experimentally analyzed and transcription factors regulating ERG20 were not described so far.In mammalian cells ergosterol biosynthesis and fatty acid biosynthesis pathways are to some extent commonly regulated (Edwards et al., 2000).We investigated if ERG20 is regulated by Rsp5p in common with genes in-volved in unsaturated fatty acid biosynthesis.It is known that transcription of the OLE1 gene encoding fatty acid desaturase is under the control of the Spt23p and Mga2p transcriptional regulators.To be active, Spt23p and Mga2p require Rsp5p-dependent ubiquitination, proteolytic processing and release from endoplasmic reticulum membranes (Hoppe et al., 2000;Shcherbik et al., 2003).To test if Spt23p affects expression of ERG20 we analyzed the level of Erg20p in strains with different SPT23 gene copy number.For this purpose we used a wild type strain transformed with vector alone or transformed with a multicopy plasmid expressing SPT23, and a strain with SPT23 deletion, all Northern analysis of RNA isolated from wt (T8-1D/1, pJK1) and rsp5-13 (TZ23/1, pJK1) strains grown in SC-ura-leu at permissive temperature followed by a shift to 37 o C for 2.5 h.Radioactive probes specific for ERG20 or ACT1 were used.Quantification of ERG20 mRNA specific signals after correction for ACT1 mRNA is shown below.
expressing ProtA-Erg20p from the pJK1 plasmid.The results presented in Fig. 3 showed that neither SPT23 deletion nor SPT23 overexpression had an effect on Erg20p level.
Because of the genetic links between RSP5 and the cell wall integrity pathway we tested if ERG20 expression is under the regulation of protein kinase C (Pkc1p), an important component of this signaling pathway.Northern analysis performed in pkc1D and wild type strain showed indeed lower expression (0.6 of wild type) of ERG20, both from the genomic copy and a copy expressed from a multicopy plasmid, when Pkc1p was absent (Fig. 4).This result indicates that ERG20 and the cell wall integrity pathway use at least one common signal.Computer analysis of the ERG20 promoter by Mat Inspector (http:// imgt.cines.fr)revealed potential binding sites for several transcription factors, including Rlm1p (Jung et al., 2002) and Gcn4p (Natarajan et al., 2001).However, our studies indicate that neither Rlm1p nor Gcn4p regulate expression of ERG20 (not shown).Since the presence of other transcription factors, besides Rlm1p, controlled by the Pkc1p-dependent pathway has been postulated (see a review by Smits et al., 2001) these so far unidentified factors might be responsible for the regulation of ERG20.

Cellular ergosterol content is low in rsp5-13 mutant
Low expression of Erg20p in rsp5-13 mutant could result in changes of the amounts of the end products of the isoprenoid pathway.Since most of farnesyl diphosphate produced by Erg20p is used for ergosterol biosynthesis, we measured the level of ergosterol in rsp5-13 cells and in wild type cells grown in YEPD at 30 o C. The amount of ergosterol was lower by about 21% in rsp5-13 cells than in wild type cells (Table 2).This difference was statistically significant.The presented results indicate that Rsp5p indirectly affects the cellular ergosterol level.
Nystatin is an antifungal drug that interacts selectively with membrane ergosterol but not with other sterols (Lees et al., 1995) and absence of ergosterol is known to confer nystatin-resistant growth.Therefore, we determined if the rsp5-13 mutation affects growth of cells in the presence of nystatin.Growth of rsp5-13 and wild type strains was tested by drop test on YEPD medium containing nystatin at 30 o C. The rsp5-13 mutant cells were slightly resistant to nystatin (Fig. 5).This result is in agreement with the lower content of ergosterol in the rsp5-13 mutant.

Cell wall is defective in rsp5-13 mutant
Since rsp5 mutant cells showed lower ERG20 expression and in consequence a decreased flux of the isoprenoid pathway, we assumed that also other products of isoprenoid pathway besides ergosterol might be synthe-sized with lower efficiency.Such products as dolichols are very important for protein glycosylation in the endoplasmic reticulum and disturbances in their synthesis cause defects of maturation and transport of cell wall proteins and in consequence in cell wall biogenesis.Therefore, we studied cell wall morphology and resistance to treatments which disturb its function.Electron microscopy was used to investigate cell wall morphology in wild type and rsp5-13 cells grown on YEPD at 30 o C. Our control cells displayed a single thin layer cell wall (Fig. 6A), but some cells (about 6%) exhibited aberrant cell wall morphology.The proportion was different in the rsp5-13 mutant as only 42% of cells   A. Cell wall structure is abnormal in rsp5-13 strain.Electron microscopy was performed on wild type (MK1) and rsp5-13 (MK5) strains grown on YEPD at 30 o C. Bar represents 1 mm.B. rsp5-13 strain is sensitive to calcofluor white.Growth of wild type (MK1) and rsp5-13 (MK5) strains on YEPD and YEPD with calcofluor white (YEPD+CFW) C. rsp5-13 strain is sensitive to zymolyase treatment similarly as wild type strain.A 600 of cell suspensions was measured at indicated time intervals after zymolyase addition.
showed normal morphology, while the cell wall of the mother cell was thicker than that of the bud in 58% of dividing cells.As shown in Fig. 6A, the structure of the thick cell wall in rsp5-13 was uniform, with no visible additional layers, but the thickness differed around the cell.
Calcofluor white is a drug which destabilizes cell wall by binding to chitin.Changes in resistance to this drug are often indicative of cell wall abnormalities and are accompanied by changes in chitin level (De Nobel et al., 2001).Growth of wild type and rsp5-13 strains was compared on YEPD containing calcofluor white.The rsp5-13 strain appeared much more sensitive than wild type to this drug (Fig. 6B) confirming that cell wall function is disturbed in rsp5-13.To find out if the sensitivity to calcofluor white is a result of abnormal call wall composition the content of chitin in the cell wall was measured.The cell wall of rsp5-13 strain contained almost twice as much chitin as the wild type (Table 3) and this might be the reason for the observed calcofluor white sensitivity.We also tested other parameters of the cell wall, the content of glucan, the main component of the cell wall, and sensitivity to zymolyase, a mixture of enzymes comprising b-1,3-glucanase and protease activities.There was only a slightly higher level of glucan content in rsp5-13 than in the wild type strain (Table 3) and the sensitivity to zymolyase was comparable in these two strains (Fig. 6C).
These results document that defects of the Rsp5 ubiquitin ligase result in thickening of the cell wall and this correlates with a higher content of chitin and increased sensitivity to a chitin-binding drug.In the same cells the b-1,3-glucan layer and sensitivity to cell wall degrading enzymes are not altered.

DISCUSSION
Regulation of the isoprenoid pathway is intensively studied because products of this pathway have great impact on cell physiology.Erg20p is a branch point enzyme of this pathway and farnesol deriving from the Erg20p product, FPP, has been shown to be a non-sterol regulator in the synthesis of ergosterol in yeast and cholesterol in human (for a review see Grabiñska & Palamarczyk, 2002).In this report we provide data that ERG20 gene expression is regulated at the level of ERG20 transcript synthesis or degradation, and that this regulation is dependent on the ubiquitin system and the cell wall integrity pathway.In particular, the Rsp5 ubiquitin protein ligase affects greatly the ERG20 transcript level.In consequence, Rsp5p affects also cellular content of ergosterol and cell wall structure and composition.
Changes in transcriptional regulation of genes in response to altered activity of Rsp5p have been reported previously.RSP5 affects transcriptional activation of heat shock element (HSE)-dependent targets (Kaida et al., 2003) and transcriptional regulation of OLE1 gene, encoding fatty acid desaturase (Hoppe et al., 2000).This latter regulation is known in detail and it involves the Spt23p and Mga2p transcriptional regulators.They are docked via transmembrane domain in the endoplasmic reticulum and must be processed and released, depending on Rsp5p, to perform their action in the nucleus ( al., 2000;Shcherbik et al., 2003;2004).However, we did not find evidence that this mechanism operates in the regulation of ERG20 expression.It has been demonstrated that Rsp5p also ubiquitinates and directs for degradation the large subunit of RNA polymerase II (Huibregtse et al., 1997;Beaudenon et al., 1999) and in this way may affect transcription of some genes.Transcriptional downregulation of ERG20 in rsp5-13 might be also a more indirect effect of changes in cellular physiology resulting from defects in Rsp5p.In theory it is also possible that Rsp5p affects ERG20 transcript stability, not the synthesis.We found a strict (three fold decrease) correlation between low ERG20 transcript level and diminished levels of Erg20p in rsp5-13.Thus, a translational regulation of ERG20 expression was probably not involved.However, Erg20p level was not quantitatively related to the sterol content, which was diminished only by about 21%, indicating for possible regulation of Erg20p at the level of enzyme activity (Szkopińska et al., 2000).Ergosterol is mainly present in the plasma membrane (Zinser et al., 1993).A regulatory role in the transport of ergosterol to this destination is played by lipid particles and sterol/steryl esters homeostasis through an esterification/hydrolysis mechanism (Mullner & Daum, 2004) so the total level of ergosterol does not necessarily reflect the plasma membrane level.Resistance of rsp5-13 to nystatin, however, indicates that the plasma membrane level of ergosterol must be lower in this mutant.Since ergosterol is necessary for several steps of endocytosis (Munn et al., 1999;Heese-Peck et al., 2002) this may possibly be an additional reason why endocytosis is impaired in rsp5 mutants (Żołądek et al., 1997;Gajewska et al., 2001).Dolichol, one of the products of the isoprenoid pathway, is used as a sugar carrier lipid or sugar donor in N-glycosylation, O-mannosylation and GPI-anchoring, all important for cell wall synthesis and secretion.The cell wall is a complex and dynamic structure which is remodeled in response to different environmental conditions like osmotic or temperature stress, nutritional factors and pheromones (Smits et al., 2001).Cell wall remodeling is regulated by the cell wall integrity pathway comprising Pkc1p and the MAP kinase cascade.We have found downregulation of ERG20 in pkc1D but it was only by about 40% suggesting that the cell wall integrity pathway is involved and also other factors contribute to the transcriptional regulation of this gene.The pkc1D dependent regulation of ERG20 was not mediated via the transcriptional factor Rlm1p and other factors must be sought.The presence of Pkc1p-dependent factors other than Rlm1p has long been suspected (Smits et al., 2001).
The yeast cell wall has a layered structure and is composed mainly of glucan (60%), mannoproteins (40%) and chitin (1%).About 90% of chitin in Saccharomyces cerevisiae is synthesized by chitin synthase 3 (Chs3p), one of the three yeast chitin synthases.Chs3p transits through the endoplasmic reticulum/Golgi secretory pathway to the plasma membrane and later is retrieved by enodocytosis.The cycling between the plasma membrane and endosomes is highly regulated by several proteins (Smits et al., 2001).Deregulation results in abnormal chitin content in the cell wall.It is apparent from recent studies that deletion of genes that influence protein glycosylation or endocytosis yields strains that have high chitin content (Bulik et al., 2003).Thus, the observed changes of cell wall structure and higher chitin content in rsp5 mutant may reflect defects in two processes: possible defects in glycosylation of cell wall proteins due to downregulation of the isoprenoid pathway and slower endocytosis affecting chitin synthase cycling.

Figure 1 .
Figure 1.The level of Erg20p is lower in rsp5-13 and depends on Mod5p.
o C for 2.5 hours.Protein extracts were analyzed by Western blot with anti-ferrochelatase antibody.Quantification of ProtA-Erg20p level after correction for ferrochelatase as a control is shown below.

Figure 2 .
Figure 2. The level of ERG20 mRNA is lower in rsp5-13 compared to wild type.

Figure 4 .
Figure 4. Transcription of ERG20 is controlled by cell wall integrity signaling pathway.Northern blot analysis of RNA isolated from wt (BY4742) and pkc1D (Y23133-1D) strains bearing vector or pLB2 grown on SC-ura at 30 o C. Radioactive probes specific for ERG20 or ACT1 were used.Quantification of ERG20 mRNA specific signals after correction for ACT1 mRNA is shown below.

Table 3 . Effect of rsp5-13 on chitin and glucan content in cell wall.
Hoppe et