Nucleic Acids Research

Yeast strains

YB427 (MATa, nmt1-451D, ura3, his3, ade2, ade3, leu2, trp1) has been described previously (7). The nmt1-451D allele in this strain was replaced by NMT1 using the integrating vector pBB395. pBB395 is a derivative of pRS305 (21) that contains a region of NMT1 extending from its nucleotide 1031 (15) to 552 bases downstream of its stop codon. pBB395 was linearized by digestion with MluI and used to transform YB427, yieldingYB553 (NMT1 and a leucine prototroph). YB579 was constructed using a similar strategy except that the AvrII-HindIII fragment containing the NMT1 domain described above was subcloned into pRS306 (21) and YB427 transformants were isolated based on their uracil prototrophy.

INO2, INO4 or OPI1 was deleted in YB427 and YB553 and replaced by HIS3 (22). To do so, the isogenic strains were first transformed with pTSV31A (kindly supplied by Alan Bender, Indiana University). pTSV31A contains URA3 and ADE3, and rescues the histidine auxotrophy produced by the ade3 allele in YB427 and YB553. The HIS3 gene in pBM2815 (obtained from Mark Johnston, Washington University) was amplified by PCR using (i) a primer encompassing 45 nt immediately upstream of the initiator Met codon of the target gene, followed by 19 bases from the region 196-177 nt upstream of the ATG of HIS3 (5[prime]-GGCCTCCTCTAGTACACTC-3[prime]) and (ii) a primer encompassing the 45 nt immediately downstream of the stop codon of the target gene followed by 19 bases from the region 154-173 nt downstream of the HIS3 stop codon (5[prime]-GCGCGCCTCGTTCAGAATG-3[prime]). The PCR product was introduced into YB427 and YB553 and transformants were isolated based on their histidine prototropy. Proper integration of HIS3 at the locus of interest was confirmed by PCR using genomic DNA as template (23) plus primer pairs that flanked the junction between the promoter region of the deleted gene and the inserted HIS3 DNA. Cells with proper integrations of HIS3 were plated on synthetic media containing 5-fluoro-orotic acid (PCR, Inc.) to expel pTSV31A. These manipulations yielded the following panel of isogenic strains: YB582 (nmt1-451D,ino2::HIS3), YB583 (NMT1,ino2::HIS3), YB585 (nmt1-451D,opi1::HIS3), YB586 (NMT1,opi1::HIS3), YB588 (nmt1-451D,ino4::HIS3), YB589 (NMT1,ino4::HIS3).

Promoter activity assays

The promoter activities of seven genes were assessed in these isogenic strains. Given the relatively large number of combinations of strains and genes to be assayed, we elected to define promoter activities using episomes containing portions of the 5[prime] non-transcribed regions of these genes linked to one of two reporters.

Plasmids containing the chloramphenicol acetyltransferase gene (cat) fused to 5[prime]-transcriptional regulatory regions from INO2, INO4, OPI1 and INO1 were generously supplied by John Lopes (Wayne State University) and are described in Ashburner and Lopes (24). Their inserts were removed by digestion with KpnI and SacI in the case of INO2-cat and OPI1-cat, or KspI and ClaI in the case of INO4-cat and INO1-cat. These restriction fragments were then inserted into KpnI/SacI- or KspI/ClaI-digested pRS316 (21). The resulting recombinant plasmids were used to transform the yeast strains listed above.

Plasmids containing Escherichia coli [beta]-galactosidase (lacZ) fused to the 5[prime] transcriptional regulatory regions of FAS1 and FAS2 (pSCFAS1 and pSCFAS2, respectively; 18) were kindly supplied by Subrahmanyam Chirala (Baylor College of Medicine). An ACC1-lacZ fusion gene was constructed by PCR amplification of the region of ACC1 bounded by its SalI and HindIII sites and then subcloning the product into YEp365 (American Type Culture Collection). The result was pBB405 which contained the 1700 nt of ACC1 located upstream of its initiator Met codon plus the first four codons of its open reading frame (ORF), fused in-frame to the lacZ ORF. The FAS1-lacZ, FAS2-lacZ and ACC1-lacZ plasmids were each introduced into isogenic strains containing nmt1-451D (YB427) or NMT1 (YB553, YB579).

A YEp-based plasmid containing FAS1, previously isolated as a high-copy suppressor of nmt1-451D (7), plus a YEp plasmid containing FAS2 (YEPFAS2; from Subrahmanyam Chirala), were also each introduced into YB427 and YB579.

YB427, YB553 and YB579 plus their derivatives, with and without these episomes, were grown at 24°C to mid-log phase (OD₆₀₀ = 0.5-1.0) in synthetic medium (Bio101) containing 2% (w/v) glucose plus 75 µM inositol and lacking either leucine or uracil. For some experiments, the medium was supplemented with 500 µM myristate (NuCheck-Prep) and/or 1% (w/v) Brij 58. Mid-log phase cells were collected from 50 ml cultures by centrifugation at 1600 g for 10 min, washed twice in 1 M sorbitol and resuspended in the same medium with no inositol or 75 µM inositol. Equivalent numbers of cells (2-5 × 10⁷/ml) were incubated at 24, 30 or 33°C for an additional 4 h. At the end of this period, cells were pelleted and resuspended in lysis buffer [100 mM Tris-HCl (pH 8), 1 mM DTT, 20% glycerol for [beta]-galactosidase assays; 0.25 M Tris-HCl (pH 7.5) for CAT assays; 250 µl of buffer/5 ml of culture]. Cell suspensions were frozen, thawed on ice and 4-(2-aminoethyl)-benzensulfonyl fluoride hydrochloride (Boehringer Mannheim) was added to a final concentration of 200 µM. Cells were then disrupted by vortexing with an equal volume of 425-600 µm glass beads (Sigma; vortexing = six cycles of 15 s each). Cellular debris was removed by centrifugation at 16 000 g for 5 min. The protein concentration of cleared lysates was measured using the BCA assay kit (Pierce). [beta]-Galactosidase activity was measured using chlorophenol red [beta]-galactopyranoside (Boehringer Mannheim) as the substrate (25). Assays were performed using 5-50 µl of cleared lysate (2.5-150 µg protein). Units of [beta]-galactosidase activity were defined as the change in absorbance at 570 nm/min/µg of protein. CAT activity was determined by the phase extraction method (26). Units of CAT activity were defined as c.p.m. in the organic phase and expressed as a percentage of total c.p.m./µg protein/h of incubation. All strains were assayed in duplicate on at least three different occasions.

RNA analysis

Mid-log phase cells from 5 ml cultures were harvested by centrifugation at 1600 g for 5 min at 4°C and washed twice with phosphate-buffered saline (PBS). Spheroplasts were prepared by first resuspending the cell pellet in 0.6 ml of cell wall digestion buffer [1 M sorbitol, 0.1 M EDTA, pH 7.4, 50 U of Zymolyase 100-T (ICN) per ml of culture (N.B. Zymolyase 100-T was activated with addition of 10 µl of 2-mercaptoethanol per ml of cell wall digestion solution)]. This mixture was then incubated at 24°C with shaking for 15 min. Spheroplasts were lysed and RNA was recovered using the Purescript RNA Isolation kit (Gentra Systems, Inc.). For northern blot analysis, RNA samples (10 µg) were fractionated by denaturing formaldehyde-agarose gel electrophoresis and transferred to GeneScreen Plus hybridization membrane (NEN Life Sciences). Blots were probed with pAS103 (CHO1; 24) and pBM659 (ACT1; obtained from Mark Johnston, Washington University). These DNAs were labeled with ³²P using the Random Primed DNA Labeling Kit (Boehringer Mannheim). Following hybridization (GeneScreen Plus protocol) and washing, the amount of bound probe was quantitated using a phosphorimaging system (Molecular Dynamics).

Statistical analysis

Comparison of means were performed using Students t-Test (Microsoft Excel version 5.0).

nmt1-451D affects INO2 and INO4 promoter activity

To determine whether expression of INO2, INO4 and/or OPI1 are affected by nmt1-451D, 506, 495 and 439 bp of their 5[prime] non-transcribed domains (respectively) were linked to a cat reporter and the fusion genes were introduced into isogenic NMT1 and nmt1-451D strains as CEN-containing episomes. Transformants were grown at 24°C to mid-log phase in synthetic medium, harvested, and equal numbers of cells incubated for 4 h at 24, 30 and 33°C in 75 µM inositol or in the absence of inositol.

Removing inositol resulted in a 5-10-fold increase in INO2-cat expression in both NMT1 and nmt1-451D cells at 24-33°C (Fig. 1A and B). However, at each temperature, the specific activity of CAT was 2-4-fold higher in nmt1-451D cells (P < 0.05; Fig. 1A and B). In contrast, expression of INO4-cat was not significantly affected by withdrawal of inositol and was 2-4-fold lower in nmt1-451D cells (P < 0.05; Fig. 1C and D). OPI1-cat expression was not significantly altered by nmt1-451D at any of the temperatures tested (Fig. 1E and F).

Figure 1. nmt451Dp-dependent changes in INO2, INO4 and OPI1 promoter activities. Isogenic NMT1 and nmt1-451D cells with INO2-cat, INO4-cat or OPI1-cat episomes were grown at 24°C to mid-log phase in synthetic media containing 75 µM inositol. Cells were resuspended in synthetic media containing 0 or 75 µM inositol, and incubated for 4 h prior to determination of cellular CAT activity. Results are plotted as the mean ± S.E.M. of three to four independent experiments, each done in duplicate. An asterisk indicates a significant difference compared to NMT1 cells (P < 0.05).

The alterations in INO2-cat promoter activity produced by nmt1-451D are corrected by exogenous myristate

The growth defect of nmt1-451D cells can be rescued by supplementing the medium with myristate (7,16). Previous studies have shown that the exogenous myristate is imported, activated by FAA1 and FAA4 to myristoyl-CoA and then utilized by nmt451Dp to increase the level of acylation of cellular N-myristoylproteins such as ADP ribosylation factors 1 and 2 (Arf1p and Arf2p; 17,20).

We compared INO2-cat expression in mid-log phase nmt1-451D cells after incubation at 24°C for 4 h in medium with or without myristic acid supplementation, with or without inositol. A supplemental dose of 500 µM myristate was chosen because earlier studies had shown that it was sufficient to correct the reduced levels of Arfp N-myristoylation at temperatures ranging from 24 to 37°C (20). Addition of myristate had no statistically significant effects on CAT activity in NMT1 cells, whether they were incubated at 24°C in the presence or absence of inositol (Fig. 2). In contrast, myristate decreased INO2-cat expression in nmt1-451D cells, grown with or without inositol, to levels that were equivalent to those in isogenic NMT1 cells (Fig. 2).

Figure 2. Exposure of nmt1-451D cells to myristate returns INO2-cat expression to levels encountered in isogenic NMT1 cells. Cells were grown at 24°C to mid-log phase in synthetic media containing 75 µM inositol. Cells were resuspended in media containing either 0 or 75 µM and either 1% Brij alone, 1% Brij plus 500 µM myristate or 1% Brij plus 500 µM palmitate. Following a 4 h incubation at 24°C, CAT activity was measured in cell lysates. Mean values ± S.E.M. of three independent experiments, each done in duplicate, are plotted. An asterisk indicates a significant difference (P < 0.05) compared to NMT1 cells.

Figure 3. Effects of nmt1-451D on expression of known inositol-responsive genes, CHO1 and INO1. Isogenic strains were grown at 24°C to mid-log phase in the presence of 75 µM inositol. Cells were resuspended in media containing 0 or 75 µM inositol, incubated for 4 h at 30°C and total RNA was isolated. (A) Cho1p mRNA levels were defined by northern blot analysis and normalized to actin (Act1p) mRNA. (B and C) A separate series of experiments where mid-log phase cells containing an INO1-cat episome were incubated for 4 h at 24-33°C prior to assaying CAT activity. Mean values ± S.E.M. obtained from three to four independent experiments, each done in duplicate, are plotted. There are no significant differences in CAT activity between NMT1 and nmt1-451D cells.

The decrease in INO2-cat expression was specific for myristate: palmitate had no significant effect (Fig. 2). PalmitoylCoA is not a substrate for Nmt1p or nmt451Dp (9,17,27). Moreover, addition of palmitate to nmt1 cells does not correct the reduced acylation of N-myristoylproteins such as Arf1p or Arf2p (17).

These findings indicate that the increased level of INO2-cat expression observed in nmt1-451D cells is likely due to reduced acylation of one or more cellular N-myristoylproteins. Ino2p, Ino4p or Opi1p are not among these proteins since none have N-terminal sequences that would allow them to be recognized as Nmt1p (or nmt451Dp) substrates.

The effects of altered INO2 and INO4 expression in nmt1-451D cells on inositol-responsive genes

The change in INO2 and INO4 promoter activities were in opposite directions. Therefore, we examined the net effect of these changes on expression of several inositol-responsive target genes.

CHO1 encodes phosphatidylserine synthase and is normally induced when inositol is absent (28). RNA blot hybridization studies revealed that the steady-state level of Cho1p mRNA was ~2-fold higher in mid-log phase nmt1-451D cells compared to NMT1 cells after they had been incubated for 4 h at 24°C in the presence or absence of inositol (Fig. 3A). Deletion of INO2 results in a marked (17-20-fold) reduction in Cho1p mRNA levels in mid-log phase nmt1-451D (and NMT1) cells after a 4 h incubation with or without inositol (Fig. 3A plus data not shown), thereby confirming that CHO1 transcription is greatly dependent upon Ino2p. Thus, the net effect of increased INO2 expression and decreased INO4 expression in nmt1-451D cells is augmented expression of one of their target genes-CHO1.

Figure 4. FAS1-lacZ expression is increased in nmt1-451D cells. (A) Rescue of growth by FAS1 but not FAS2. NMT1 and nmt1-451D strains, containing high copy YEp plasmids with either FAS1 or FAS2 inserts, were grown overnight at 24°C in synthetic medium lacking leucine and containing 75 µM inositol. An equal number of cells were then plated on the same medium and incubated at 24 or 30°C for 3 days. (B-G) Isogenic NMT1 and nmt1-451D cells containing FAS1-lacZ, FAS2-lacZ or ACC1-lacZ episomes were grown at 24°C to mid-log phase in synthetic media containing 75 µM inositol, resuspended in synthetic media containing 0 or 75 µM inositol, incubated for 4 h at the indicated temperature and then assayed for cellular [beta]-galactosidase activity. Results are plotted as the mean ± S.E.M. of three to nine independent experiments, each done in duplicate. The asterisk indicates a significant difference compared to NMT1 cells (P < 0.05).

The changes in INO2 and INO4 promoter activities observed in nmt1-451D cells is not associated with an increase in expression of all inositol-responsive genes. INO1 encodes inositol-1-phosphate synthase, issensitive to inositol and is tightly regulated by Ino2p and Ino4p (5,6,29). INO1-cat expression in NMT1 and nmt1-451D cells was assayed using the same conditions described above for INO2-cat. The nmt1-451D allele had no significant effect on INO1 promoter activity whether cells were incubated with or without inositol (Fig. 3B and C).

FAS1 and FAS2 encode the [beta]- and [alpha]-subunits of the [alpha]₆[beta]₆ Fas complex. ACC1 encodes acetylCoA carboxylase which produces the malonyl-CoA substrate used by Fas. All three genes have ICREs in their 5[prime] non-transcribed domains (18,19,30,31). MyristoylCoA is one of the products of Fas and is normally a rare cellular acylCoA species (32). Changes in FAS1 expression in NMT1 cells are known to affect expression of FAS2 but not vice versa: i.e. deletion of FAS1 results in a decrease in Fas2p mRNA levels while deletion of FAS2 has no detectable effect on steady state Fas1p mRNA concentrations (33). We found that transformation of nmt1-451D cells with a high-copy YEp plasmid containing FAS1 rescues growth at 30 and 33°C. In contrast, transformation of the same cells with a YEp plasmid containing FAS2 does not rescue growth (Fig. 4A). Control experiments using a NMT1 strain with a well characterized mutant FAS2 allele (fas2-38; 34), demonstrated that the FAS2 episome rescues the fatty acid auxotrophy (data not shown), confirming that the plasmid produced a functional product. Together, these observations suggest that regulation of FAS1 expression is a critical determinant of overall Fas activity in nmt1-451D cells and augmented Fas expression can rescue the kinetic defects of nmt451Dp at elevated temperatures.

To determine the net effect of the altered INO2 and INO4 promoter activities in nmt1-451D cells on FAS and ACC1 expression, FAS1-lacZ, FAS2-lacZ and ACC1-lacZ episomes were introduced into the isogenic NMT1 and nmt1-451D strains. [beta]-Galactosidase activity was measured in mid-log phase cells harvested after a 4 h incubation at 24, 30 or 33°C in the presence or absence of inositol. FAS1-lacZ expression was significantly higher in nmt1-451D cells at all temperatures tested, both in the presence or absence of inositol (Fig. 4B and C). In contrast, FAS2-lacZ expression did not differ significantly between strains at any temperature, with or without exposure to inositol (Fig. 4D and E). ACC1-lacZ expression was also similar in both strains with one exception: a slight but statistically significant increase in reporter activity was noted in nmt1-451D compared to NMT1 cells at 30°C in the absence of inositol (Fig. 4F and G). Thus, augmented INO2 and reduced INO4 expression are associated with a net increase in FAS1 promoter activity.

Evidence that FAS expression in nmt1-451D cells is regulated by factors other than INO2 and INO4

FAS1-lacZ and FAS2-lacZ activities were compared in isogenic NMT1 and nmt1-451D strains with wild type and null alleles of INO2, INO4 or OPI1. Cells were grown at 24°C in synthetic media containing 75 µM inositol to mid-log phase, transferred to fresh media (with inositol) and then incubated at 24, 30 or 33°C for an additional 4 h before reporter activity was measured in cell lysates.

In NMT1 cells, deletion of INO2 results in a significant decrease in the expression of FAS1-lacZ (and FAS2-lacZ ) at 24, 30 and 33°C (Fig. 5A and B). Remarkably, although deletion of INO2 results in a decrease in FAS1-lacZ (and FAS2-lacZ)expression in nmt1-451D cells at 24°C, when the temperature is increased to 30 or 33°C this diminished expression disappears (Fig. 5C and D). Thus, it appears that nmt1-451Dino2[Delta]cells are able to compensate for the loss of Ino2p and maintain FAS1 promoter activity at augmented levels in response to deficiencies in protein N-myristoylation.

Figure 5. FAS1 and FAS2 promoter activities in NMT1 and nmt1-451D cells in the presence or absence of Ino2p, Ino4p or Opi1p. Expression of FAS1-lacZ and FAS2-lacZ was measured as described in Figure 4. Results are presented as the change in reporter expression relative to the NMT1 or nmt1-451D parental strain lacking the gene deletion. Values represent the mean ± S.E.M. of three independent experiments each performed in duplicate. An asterisk indicates a significant difference compared to the parental strain (P < 0.05).

Figure 6. Effect of deleting INO2, INO4 or OPI1 on growth of NMT1 and nmt1-451D cells. Strains were grown to mid-log phase at 24°C in synthetic media containing 75 µM inositol. An equal number of cells were transferred to plates of synthetic media either lacking inositol or containing 75 µM inositol and incubated at the indicated temperature for 3 days.

This compensation is not due to a loss of dependency on Ino4p in nmt1-451D cells. In NMT1ino4[Delta]cells, loss of Ino4p produces a decrease in FAS1-lacZ and FAS2-lacZ expression similar to that observed with loss of Ino2p (Fig. 5A and B). Moreover, deletion of INO4 in nmt1-451D cells results in an even greater decrease in FAS1 and FAS2 promoter activity than in NMT1 cells (Fig. 5A-D). The compensation is also not due to a loss of sensitivity to Opi1p repression. Deletion of OPI1 causes a significant increase in FAS1-lacZ (but not FAS2-lacZ) expression at 30 and 33°C. The increase is similar in NMT1 and nmt1-451D cells (Fig. 5A-D).

Together, these results suggest that FAS1 expression is augmented in nmt1-451D cells in response to undermyristoylation of one or more cellular N-myristoylproteins through increased expression of INO2 and increased activity of an unknown transcriptional activator (or activators) that can substitute for Ino2p and/or compensate for reduced expression of INO4.

To further assess the relative importance of Ino2p, Ino4p, Opi1p and the as yet unknown myristoylation-sensitive transcription factor(s) (MSTF) to nmt1-451D cells, we compared the growth of NMT1 and nmt1-451D strains containing a wild type or null allele of INO2, INO4 or OPI1. Each of the eight isogenic strains was allowed to grow at 24°C to mid-log phase on synthetic medium containing 75 µM inositol, after which time equal numbers of cells were plated on synthetic medium with or without inositol. NMT1 and nmt451D cells containing either ino2[Delta]or ino4[Delta]should be inositol auxotrophs and as expected, they fail to grow without inositol (Fig. 6A). In addition, removal of the Opi1p repressor produces no further benefit to the rescue of nmt1-451D cells produced by inositol withdrawal. In medium containing 75 µM inositol, removal of the Opi1p repressor rescues growth of nmt1-451D cells at 30 and 33°C (Fig. 6B)-a result consistent with the finding that opi1[Delta]causes a significant increase FAS1 promoter activity under these conditions (see above). Removal of Ino2p does not rescue growth of nmt1-451D cells at 30 or 33°C in medium containing inositol (Fig. 6B). This result suggests that the unknown MSTF(s) cannot fully substitute for Ino2p and maintain the viability of this strain under these conditions. Finally, growth of nmt1-451D cells at 30°C is rescued by ino4[Delta](Fig. 6B). This finding indicates that further reductions in the levels of INO4 expression below those already manifested by nmt1-451D cells may be beneficial.

Figure 7. Working model of the relationship between protein N-myristoylation and regulation of Ino4p-dependent gene transcription. In wild type cells, Ino4p is known to partner with Ino2p to help maintain FAS expression and to autoregulate INO2 transcription. Ino4p does not form a homodimer and apparently cannot act as a transcriptional activator without a partner (4). Autoregulation of INO4 in wild type cells is INO2 independent, suggesting the existence of another as yet unknown protein partner (24), termed factor X in the Figure. Conditions leading to a reduction in N-myristoylation of one or more cellular N-myristoylproteins result in an increase in INO2 and FAS transcription. We hypothesize that Ino4p forms a heterodimer with MSTFs and that this Ino4p:MSTF heterodimer has limited functional overlap with the Ino2p:Ino4p heterodimer. The overlap includes the ability to support FAS transcription (even in ino2[Delta]cells) and activate INO2 transcription. Undermyristoylation of cellular proteins is also associated with a reduction in INO4 promoter activity. MSTF:Ino4p heterodimers may have reduced ability to activate INO4 or MSTF may block the ability of Factor X to interact with Ino4p and autoregulate INO4. Alternatively, MSTF(s) may be N-myristoylated and its (their) function regulated by the presence or absence of a myristoyl moiety-raising the possibility that MSTF is Factor X.

DISCUSSION

We have found that the promoter activities of INO2 and INO4 are sensitive to the state of protein N-myristoylation. The activity of INO2 is significantly higher, and INO4 significantly lower, in nmt1-451D compared to NMT1 cells. The result is a net increase in expression of some inositol target genes, including FAS1. Augmented expression of FAS1 is likely to help overcome the kinetic defects in nmt451Dp by providing more myristoylCoA substrate. FAS1 expression is Ino2p-dependent in NMT1 cells. Remarkably, FAS1 expression is not reduced by deletion of INO2 in nmt1-451D cells as incubation temperatures are increased to 30-33°C and efficient acylation of cellular N-myristoylproteins is threatened. The ability to maintain expression of FAS1 in nmt1-451D cells that lack Ino2p suggests the existence of another transcription factor, or factors, whose expression/activity is (are) inversely related to the overall levels of cellular protein N-myristoylation.

We hypothesize that reduced acylation of one or more cellular N-myristoylproteins results in a compensatory increase in activity of this postulated transcription factor, or factors, which can substitute for Ino2p and maintain relatively high levels of FAS1 expression. Expression of INO2 is autoregulated: Ino2p is required to maintain INO2 transcription through a mechanism that requires INO4. INO4 is also autoregulated but through a mechanism that is INO2-independent (24). We do not know whether the myristoylation-sensitive transcription factor(s) (MSTF) is only expressed in the absence of Ino2p, whether it is capable of functioning together with Ino4p, whether it contributes to the increased INO2 and decreased INO4 promoter activities observed in nmt1-451D cells, whether its expression is directly regulated through an inositol-responsive pathway, and whether it acts through an UAS_INO.

We do know that while MSTF appears to be able to fulfill the role of INO2 in regulating FAS expression in nmt1-451D cells, they must not be functionally equivalent since other inositol-responsive genes maintain INO2-dependent expression at 30-33°C. For example, deletion of INO2 causes a loss in CHO1 expression as acylation of N-myristoylproteins in nmt1-451D cells is diminished.Moreover, nmt1-451D cells devoid of Ino2p exhibit inositol auxotrophy at 24-33°C, indicating that INO1 is not expressed at sufficient levels.

The identity of this factor or factors remain(s) unclear. Our findings suggest a working model where Ino4p may have more than one partner (Ino2p and MSTF; Fig. 7). Attempts to demonstrate interactions between each of the known S.cerevisiae bHLH proteins and Ino4p have failed to date, except in the case of Ino2p (John Lopes, personal communication). A number of regulatory proteins that do not belong to the bHLH family are known to affect transcriptional activation and repression of inositol-regulated genes. RAP1, ABF1 and REB1 are required for maximal expression of FAS1 and FAS2 (35). SIN3 is required for maximal repression of INO1 (36). UME6 and DEP1 affect repression and activation of phospholipid biosynthetic genes (37,38). The effect of nmt1-451D on their expression has not been defined. None of their protein products has an N-terminal Gly and therefore they cannot be Nmt1p substrates. These factors are unlikely to be the unknown transcriptional activator suggested by our studies: their mutational inactivation generally results in pleiotropic deficiencies in gene activation and repression, rather than the selective effects observed in nmt1-451D cells. Identification of the myristoylation-sensitive transcription factor or factors should provide molecular details about how the state of protein N-myristoylation in cells serves to influence expression of inositol-responsive genes, including INO2 and INO4.

ACKNOWLEDGEMENTS

We thank John Lopes for generously providing reagents and for helpful comments during the course of this work. This work was supported in part by grants from the National Institutes of Health (AI38200) and the Monsanto Company.

REFERENCES

1. Greenberg, M.L. and Lopes, J.M. (1996) Microbiol. Rev., 60, 1-20. MEDLINE Abstract

2. Bachhawat, N., Ouyang, Q. and Henry, S.A. (1995) J. Biol. Chem., 270, 25087-25095. MEDLINE Abstract

3. Nikoloff, D.M. and Henry, S.A. (1994) J. Biol. Chem., 269, 7402-7411. MEDLINE Abstract

4. Schwank, S., Ebbert, R., Raufenstrauß, K., Schweizer, E. and Schüller, H.-J. (1995) Nucleic Acids Res., 23, 230-237. MEDLINE Abstract

5. White, M.J., Hirsch, J.P. and Henry, S.A. (1991) J. Biol. Chem., 266, 863-872. MEDLINE Abstract

6. Ashburner, B.P. and Lopes, J.M. (1995) Proc. Natl. Acad. Sci. USA, 92, 9722-9726. MEDLINE Abstract

7. Johnson, D.R., Cok, S.J., Feldman, H. and Gordon, J.I. (1994) Proc. Natl. Acad. Sci. USA, 91, 10158-10162. MEDLINE Abstract

8. Johnson, D.R., Knoll, L.J., Levin, D.E. and Gordon, J.I. (1994) J. Cell Biol., 127, 751-762. MEDLINE Abstract

9. Bhatnagar, R.S., Schall, O.F., Jackson-Machelski, E., Sikorski, J.A., Devadas, B., Gokel, G.W. and Gordon, J.I. (1997) Biochemistry, 36, 6700-6708. MEDLINE Abstract

10. Rudnick, D.A., McWherter, C.A., Rocque, W.J., Lennon, P.J., Getman, D.P. and Gordon, J.I. (1991) J. Biol. Chem., 266, 9732-9739. MEDLINE Abstract

11. Weston, S.A., Camble, R., Colls, J., Rosenbrock, G., Taylor, I., Egerton, M., Tucker, A.D., Tunnicliffe, A., Mistry, A., Mancia, F., de la Fortelle, E., Irwin, J., Bricogne, G. and Pauptit, R.A. (1998) Nature Struct. Biol., 5, 213-221.

12. Towler, D.A., Adams, S.P., Eubanks, S.R., Towery, D.S.,Jackson-Machelski, E., Glaser, L. and Gordon, J.I. (1987)Proc. Natl. Acad. Sci. USA, 84, 2708-2712. MEDLINE Abstract

13. Towler, D.A., Adams, S.P., Eubanks, S.R., Towery, D.S.,Jackson-Machelski, E., Glaser, L. and Gordon, J.I. (1988)J. Biol. Chem., 263, 1784-1790. MEDLINE Abstract

14. Duronio, R.J., Rudnick, D.A., Adams, S.P., Towler, D.A. and Gordon, J.I. (1991) J. Biol. Chem., 266, 10498-10504. MEDLINE Abstract

15. Duronio, R.J., Towler, D.A., Heuckeroth, R.O. and Gordon, J.I. (1989) Science, 243, 796-800. MEDLINE Abstract

16. Duronio, R.J., Rudnick, D.A., Johnson, R.J., Johnson, D.R. and Gordon, J.I. (1991) J. Cell Biol., 113, 1313-1330. MEDLINE Abstract

17. Zhang, L., Jackson-Machelski, E. and Gordon, J.I. (1996) J. Biol. Chem., 271, 33131-33140. MEDLINE Abstract

18. Chirala, S.S. (1992) Proc. Natl. Acad. Sci. USA, 89, 10232-10236. MEDLINE Abstract

19. Chirala, S.S., Zhong, Q., Huang, W. and Al-Feel, W. (1994)Nucleic Acids Res., 22, 412-418. MEDLINE Abstract

20. Lodge, J.K., Jackson-Machelski, E., Devadas, B., Zupec, M.E., Getman, D.P., Kishore, N., Freeman, S.K., McWherter, C.A., Sikorski, J.A. and Gordon, J.I. (1997) Microbiology, 143, 357-366. MEDLINE Abstract

21. Sikorski, R. S. and Hieter, P. (1989) Genetics, 122, 19-27. MEDLINE Abstract

22. Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F. and Cullin, C. (1993) Nucleic Acids Res., 21, 3329-3330. MEDLINE Abstract

23. Hoffman, C.S. and Winston, F. (1987) Gene, 57, 267-272. MEDLINE Abstract

24. Ashburner, B.P. and Lopes, J.M. (1995) Mol. Cell. Biol., 15, 1709-1715. MEDLINE Abstract

25. Eustice, D.C., Feldman, P.A., Colberg-Poley, A.M., Buckery,R.M. and Neubauer, R.H. (1991) Biotechniques, 11, 739-742. MEDLINE Abstract

26. Seed, B. and Sheen, J.-Y. (1988) Gene, 67, 271-277. MEDLINE Abstract

27. Rudnick, D.A., Rocque, W.J., McWherter, C.A., Toth, M.V.,Jackson-Machelski, E. and Gordon, J.I. (1993)Proc. Natl. Acad. Sci. USA, 90, 1087-1091. MEDLINE Abstract

28. Bailis, A.M., Lopes, J.M., Kohlwein, S.D. and Henry, S.A. (1992)Nucleic Acids Res., 20, 1411-1418. MEDLINE Abstract

29. Hirsch, J.P. and Henry, S.A. (1986) Mol. Cell. Biol., 6, 3320-3328. MEDLINE Abstract

30. Hasslacher, M., Ivessa, A.S., Paltauf, F. and Kohlwein, S.D. (1993)J. Biol. Chem., 268, 10946-10952. MEDLINE Abstract

31. Schüller, H.-J., Hahn, A., Tröster, F., Schütz, A. and Schweizer, E. (1992b) EMBO J., 11, 107-114. MEDLINE Abstract

32. Schjerling, C.K., Hummel, R., Hansen, J.K., Borsting, C., Mikkelsen, J.M., Kristiansen, K., Knudsen, J. (1996) J. Biol. Chem., 271, 22514-22521. MEDLINE Abstract

33. Schüller, H.-J., Förtsch, B., Rautenstrauss, B., Wolf, D.H., Schweizer, E. (1992) Eur. J. Biochem. 203, 607-614. MEDLINE Abstract

34. Schweizer, E., Werkmeister, K. and Jain, J. (1978) Mol. Cell. Biochem., 21, 95-107. MEDLINE Abstract

35. Schüller, H.-J., Schütz, A., Knab, S., Hoffmann, B. and Schweizer, E. (1994) Eur. J. Biochem., 225, 213-222. MEDLINE Abstract

36. Slekar, K.H. and Henry, S.A. (1995) Nucleic Acids Res., 23, 1964-1969. MEDLINE Abstract

37. Jackson, J.C. and Lopes, J.M. (1996) Nuceic Acid Res., 24, 1322-1329.

38. Lamping, E., Lückl, J., Paltauf, F., Henry, S.A. and Kohlwein, S.D. (1995) Genetics, 137, 55-65.

---

*To whom correspondence should be addressed. Tel: +1 314 362 7243; Fax: +1 314 362 7047; Email: jgordon@pharmdec.wustl.edu

---

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 4 Jun 1998
Copyright©Oxford University Press, 1998.
CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				K. A. Robinson and J. M. Lopes SURVEY AND SUMMARY: Saccharomyces cerevisiae basic helix-loop-helix proteins regulate diverse biological processes Nucleic Acids Res., April 1, 2000; 28(7): 1499 - 1505. [Abstract] [Full Text] [PDF]

				J. A. Graves and S. A. Henry Regulation of the Yeast INO1 Gene: The Products of the INO2, INO4 and OPI1 Regulatory Genes Are Not Required for Repression in Response to Inositol Genetics, April 1, 2000; 154(4): 1485 - 1495. [Abstract] [Full Text]

				L. Block-Alper, P. Webster, X. Zhou, L. Supekova, W. H. Wong, P. G. Schultz, and D. I. Meyer IN02, A Positive Regulator of Lipid Biosynthesis, Is Essential for the Formation of Inducible Membranes in Yeast Mol. Biol. Cell, January 1, 2002; 13(1): 40 - 51. [Abstract] [Full Text] [PDF]

This Article Abstract Print PDF (240K) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (5) Request Permissions Commercial Re-use Guidelines for Open Access NAR Content Google Scholar Articles by Cok, S. J. Articles by Gordon, J. I. Search for Related Content PubMed PubMed Citation Articles by Cok, S. J. Articles by Gordon, J. I. Social Bookmarking          What's this?