Characterization of the ORF YBR264c in Saccharomyces cerevisiae, which encodes a new yeast Ypt that is degraded by a proteasome-dependent mechanism

We identified the ORF YBR264c during the systematic sequencing of the Saccharomyces cerevisiae genome. It encodes a putative protein of 218 amino acids. We demonstrate here that the gene is indeed expressed and encodes a new Ypt in yeast. This protein specifically binds guanine nucleotides and interacts via its C-terminal end with the unique Rab GDP Dissociation Inhibitor (RabGDI). In accordance with a recent proposal, the gene is now designated YPT10. No mutant phenotype could be associated with inactivation of the gene. However, overexpression of YPT10 resulted in defects in growth; microscopic examination of such cells revealed an overabundance of vesicular and tubular structures, suggesting some alteration in the function of the Golgi apparatus. In addition, degradation of the Ypt10 protein, which possesses a PEST sequence, is shown to be dependent on proteasome activity.     

Functional implications of genetic interactions between genes encoding small GTPases involved in vesicular transport in yeast

Ras-related, guanine nucleotide-binding proteins of the Ypt/Rab family play a key role at defined steps in vesicular transport, both in yeast and in mammalian cells. In yeast, Ypt1p has an essential function late in endoplasmic reticulum (ER) to Golgi transport, and the redundant Ypt31/Ypt32 GTPases have been proposed to act in transport through and/or from the Golgi. Here we report that mutant alleles of YPT31 and YPT32, whose gene products have a reduced affinity for GTP, are able to suppress the dominant lethal phenotype of YPT1 ^N121I . Co-expression of YPT1 ^N121I and the suppressor YPT31 ^N126I allow essentially undisturbed secretory transport in the absence of the respective wild-type GTPases. Such mutant cells massively overaccumulate 60–100 nm vesicles and are heat sensitive. It appears likely that the mutant GTPases, which are defective in nucleotide binding, compete for the binding of common interacting protein(s). These and other genetic interactions between YPT1, YPT31/32, ARF1 and SEC4 described here strongly support the view that Ypt31p and Ypt32p have a central, Golgi-associated function in anterograde or retrograde transport. Received: 28 August 1998 / Accepted: 14 October 1998     

Mutational analysis of Vam4/Ypt7p function in the vacuolar biogenesis and morphogenesis in the yeast,Saccharomyces cerevisiae

Summary The vacuole is one of the most prominent compartments in yeast cells. The wild-type yeast cells have a large vacuolar compartment which occupies approximately a quarter of the cell volume, while thevam4 mutant cells exhibit highly fragmented vacuolar morphology. We isolated theVAM4 gene and found that theVAM4 is identical to theYPT7 which encodes a member of small GTP-binding protein superfamily. We introduced mutations to theVAM4/YPT7 which alter nucleotide binding characteristics of the gene product specifically, and their activities for the vacuolar morphogenesis were examined by transforming the mutant genes into yeast cells. The Thr22Asn mutation, which was expected to fix the protein in the GDP-bound state, resulted in loss of function in the vacuolar morphogenesis. Subcellular fractionation analysis indicated that the mutant molecule did not associate with intracellular membranes efficiently. In contrast, Vam4/Ypt7p with the Gln68Leu mutation, which was expected to be the GTP-bound form, complemented the fragmented vacuolar morphology of vam4 mutant cells. Vam4/Ypt7p with the Gln68Leu mutation also complemented the defects in the biogenesis of vacuolar alkaline phosphatase whose maturation requires the proper function of Vam4/Ypt7p. Overexpression of the mutant proteins in wild-type cells did not develop dominant-negative effects on the vacuolar assembly. These results indicated that the GTP-bound form of Vam4/Ypt7p promotes the biogenesis and morphogenesis of the yeast vacuolar compartment.Abbreviations ALP alkaline phosphatase - CDE centromeric - DNA element - CPY carboxypeptidase Y - GST glutathione S-transferase     

Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p).

From the budding yeast S. cerevisiae, we have cloned a gene, YPT7, that encodes a GTP-binding protein belonging to the Ypt family of ras-related proteins. The 208 amino acid protein shares identical effector domain and C-terminal sequences with the mammalian Rab7 protein. YPT7 gene disruption did not impair cellular growth at temperatures ranging from 17 degrees C to 37 degrees C. ypt7 null mutants are characterized by highly fragmented vacuoles and differential defects of vacuolar protein transport and maturation. The uptake of alpha factor pheromone by wild-type and Ypt7p-deficient cells was found to be indistinguishable, but in mutant cells lacking Ypt7p, degradation of the endocytosed pheromone was severely inhibited. Our findings suggest a role of Ypt7p in protein transport between endosome-like compartments.     

GTP Hydrolysis Is Not Important for Ypt1 GTPase Function in Vesicular Transport

GTPases of the Ypt/Rab family play a key role in the regulation of vesicular transport. Their ability to cycle between the GTP- and the GDP-bound forms is thought to be crucial for their function. Conversion from the GTP- to the GDP-bound form is achieved by a weak endogenous GTPase activity, which can be stimulated by a GTPase-activating protein (GAP). Current models suggest that GTP hydrolysis and GAP activity are essential for vesicle fusion with the acceptor compartment or for timing membrane fusion. To test this idea, we inactivated the GTPase activity of Ypt1p by using the Q67L mutation, which targets a conserved residue that helps catalyze GTP hydrolysis in Ras. We demonstrate that the mutant Ypt1-Q67L protein is severely impaired in its ability to hydrolyze GTP both in the absence and in the presence of GAP and consequently is restricted mostly to the GTP-bound form. Surprisingly, a strain with ypt1-Q67L as the only YPT1 gene in the cell has no observable growth phenotypes at temperatures ranging from 14 to 37°C. In addition, these mutant cells exhibit normal rates of secretion and normal membrane morphology as determined by electron microscopy. Furthermore, the ypt1-Q67L allele does not exhibit dominant phenotypes in cell growth and secretion when overexpressed. Together, these results lead us to suggest that, contrary to current models for Ypt/Rab function, GTP hydrolysis is not essential either for Ypt1p-mediated vesicular transport or as a timer to turn off Ypt1p-mediated membrane fusion but only for recycling of Ypt1p between compartments. Finally, the ypt1-Q67L allele, like the wild type, is inhibited by dominant nucleotide-free YPT1 mutations. Such mutations are thought to exert their dominant phenotype by sequestration of the guanine nucleotide exchange factor (GNEF). These results suggest that the function of Ypt1p in vesicular transport requires not only the GTP-bound form of the protein but also the interaction of Ypt1p with its GNEF.     

Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p

Cells lacking the GTPase Ypt6p have defects in intracellular traffic and are temperature sensitive. Their growth is severely impaired by additional mutation of IMH1, which encodes a non-essential Golgi-associated coiled-coil protein. A screen for mutants that, like ypt6, specifically impair the growth of imh1 cells led to the identification of RIC1. Ric1p forms a tight complex with a previously uncharacterized protein, Rgp1p. The Ric1p-Rgp1p complex binds Ypt6p in a nucleotide-dependent manner, and purified Ric1p-Rgp1 stimulates guanine nucleotide exchange on Ypt6p in vitro. Deletion of RIC1 or RGP1, like that of YPT6, blocks the recycling of the exocytic SNARE Snc1p from early endosomes to the Golgi and causes temperature-sensitive growth, but this defect can be relieved by overexpression of YPT6. Ric1p largely colocalizes with the late Golgi marker Sec7p. Ypt6p shows a similar distribution, but this is altered when RIC1 or RGP1 is mutated. We infer that the Ric1p-Rgp1p complex serves to activate Ypt6p on Golgi membranes by nucleotide exchange, and that this is required for efficient fusion of endosome-derived vesicles with the Golgi.     

Two New Ypt GTPases Are Required for Exit From the Yeast trans-Golgi Compartment

Small GTPases of the Ypt/rab family are involved in the regulation of vesicular transport. These GTPases apparently function during the targeting of vesicles to the acceptor compartment. Two members of the Ypt/rab family, Ypt1p and Sec4p, have been shown to regulate early and late steps of the yeast exocytic pathway, respectively. Here we tested the role of two newly identified GTPases, Ypt31p and Ypt32p. These two proteins share 81% identity and 90% similarity, and belong to the same protein subfamily as Ypt1p and Sec4p. Yeast cells can tolerate deletion of either the YPT31 or the YPT32 gene, but not both. These observations suggest that Ypt31p and Ypt32p perform identical or overlapping functions. Cells deleted for the YPT31 gene and carrying a conditional ypt32 mutation exhibit protein transport defects in the late exocytic pathway, but not in vacuolar protein sorting. The ypt31/ 32 mutant secretory defect is clearly downstream from that displayed by a ypt1 mutant and is similar to that of sec4 mutant cells. However, electron microscopy revealed that while sec4 mutant cells accumulate secretory vesicles, ypt31/32 mutant cells accumulate aberrant Golgi structures. The ypt31/32 phenotype is epistatic to that of a sec1 mutant, which accumulates secretory vesicles. Together, these results indicate that the Ypt31/32p GTPases are required for a step that occurs in the transGolgi compartment, between the reactions regulated by Ypt1p and Sec4p. This step might involve budding of vesicles from the trans-Golgi. Alternatively, Ypt31/ 32p might promote secretion indirectly, by allowing fusion of recycling vesicles with the trans-Golgi compartment.     

Ypt32 recruits the Sec4p guanine nucleotide exchange factor,Sec2p,to secretory vesicles; evidence for a Rab cascade in yeast

SEC2 is an essential gene required for polarized growth of the yeast Saccharomyces cerevisiae. It encodes a protein of 759 amino acids that functions as a guanine nucleotide exchange factor for the small GTPase Sec4p, a regulator of Golgi to plasma membrane transport. Activation of Sec4p by Sec2p is needed for polarized transport of vesicles to exocytic sites. Temperature-sensitive (ts) mutations in sec2 and sec4 result in a tight block in secretion and the accumulation of secretory vesicles randomly distributed in the cell. The proper localization of Sec2p to secretory vesicles is essential for its function and is largely independent of Sec4p. Although the ts mutation sec2-78 does not affect nucleotide exchange activity, the protein is mislocalized. Here we present evidence that Ypt31/32p, members of Rab family of GTPases, regulate Sec2p function. First, YPT31/YPT32 suppress the sec2-78 mutation. Second, overexpression of Ypt31/32p restores localization of Sec2-78p. Third, Ypt32p and Sec2p interact biochemically, but Sec2p has no exchange activity on Ypt32p. We propose that Ypt32p and Sec4p act as part of a signaling cascade in which Ypt32p recruits Sec2p to secretory vesicles; once on the vesicle, Sec2p activates Sec4p, enabling the polarized transport of vesicles to the plasma membrane.     

Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations

cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport. None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation. On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptVlp and YptVlp are the closest algal analogs of yeast Yptlp, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Yptlp, and its effector domain resembles that of Sec4p. To define more precisely the regions that supply Yptlp function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptVlp (complementing) and YptV3p (non-complementing). Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptVlp could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation. Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins.     

Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast

The small GTPase rab5 has been shown to represent a key regulator in the endocytic pathway of mammalian cells. Using a PCR approach to identify rab5 homologs in Saccharomyces cerevisiae, two genes encoding proteins with 54 and 52% identity to rab5, YPT51 and YPT53 have been identified. Sequencing of the yeast chromosome XI has revealed a third rab5-like gene, YPT52, whose protein product exhibits a similar identity to rab5 and the other two YPT gene products. In addition to the high degree of identity/homology shared between rab5 and Ypt51p, Ypt52p, and Ypt53p, evidence for functional homology between the mammalian and yeast proteins is provided by phenotypic characterization of single, double, and triple deletion mutants. Endocytic delivery to the vacuole of two markers, lucifer yellow CH (LY) and alpha-factor, was inhibited in delta ypt51 mutants and aggravated in the double ypt51ypt52 and triple ypt51ypt52ypt53 mutants, suggesting a requirement for these small GTPases in endocytic membrane traffic. In addition to these defects, the here described ypt mutants displayed a number of other phenotypes reminiscent of some vacuolar protein sorting (vps) mutants, including a differential delay in growth and vacuolar protein maturation, partial missorting of a soluble vacuolar hydrolase, and alterations in vacuole acidification and morphology. In fact, vps21 represents a mutant allele of YPT51 (Emr, S., personal communication). Altogether, these data suggest that Ypt51p, Ypt52p, and Ypt53p are required for transport in the endocytic pathway and for correct sorting of vacuolar hydrolases suggesting a possible intersection of the endocytic with the vacuolar sorting pathway.     

Identification and structure of four yeast genes (SLY) that are able to suppress the functional loss of YPT1, a member of the RAS superfamily.

In Saccharomyces cerevisiae, the GTP-binding Ypt1 protein (Ypt1p) is essential for endoplasmic reticulum-to-Golgi protein transport. By exploiting a GAL10-YPT1 fusion to regulate YPT1 expression, three multicopy suppressors, SLY2, SLY12, and SLY41, and a single-copy suppressor, SLY1-20, that allowed YPT1-independent growth were isolated. Wild-type Sly1p is hydrophilic, is essential for cell viability, and differs from Sly1-20p by a single amino acid. SLY2 and SLY12 encode proteins with hydrophobic tails similar to synaptobrevins, integral membrane proteins of synaptic vesicles in higher eucaryotes. Sly41p is hydrophobic and exhibits sequence similarities with the chloroplast phosphate translocator. SLY12 but not SLY41 is an essential gene. The SLY2 null mutant is cold and heat sensitive. The SLY gene products may comprise elements of the protein transport machinery.     

Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport

Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.     

Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high α-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum–derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in α-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.     

TRAPP stimulates guanine nucleotide exchange on Ypt1p

TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.     

Complex formation with Ypt11p,a rab-type small GTPase,is essential to facilitate the function of Myo2p,a class V myosin,in mitochondrial distribution in Saccharomyces cerevisiae

We identified Ypt11p, a rab-type small GTPase, by its functional and two-hybrid interaction with Myo2p, a class V myosin of the budding yeast Saccharomyces cerevisiae. The tail domain of Myo2p was coimmunoprecipitated with Ypt11p, suggesting that Ypt11p forms a complex with Myo2p at its tail domain in vivo. Mutational analysis of YPT11 suggests that Myo2p is a putative effector of Ypt11p. Deletion of YPT11 induced partial delay of mitochondrial transmission to the bud, and overexpression of YPT11 resulted in mitochondrial accumulation in the bud, indicating that Ypt11p acts positively on mitochondrial distribution toward the bud. We isolated two myo2 mutants, myo2-338 and myo2-573, which showed genetic interactions with YPT11. The myo2-573 mutation, identified by a synthetic lethal interaction with ypt11-null, induced a defect in mitochondrial distribution toward the bud, indicating that Myo2p plays a crucial role in polarized distribution of mitochondria. The myo2-338 mutation was identified as the mutation that abolished the effect of overexpressed YPT11, such as the Ypt11p-dependent accumulation of mitochondria in the bud, and the affinity of Myo2p for Ypt11p was reduced. These results indicate that complex formation of Ypt11p with Myo2p accelerates the function of Myo2p for mitochondrial distribution toward the bud.     

Two GTPase isoforms, Ypt31p and Ypt32p, are essential for Golgi function in yeast.

In eukaryotic cells, monomeric GTPases of the Ypt/Rab family function as regulators at defined steps of vesicular transport in exo- and endocytosis. Here we report on the isolation and characterization of two genes (YPT31 and YPT32) of the yeast Saccharomyces cerevisiae which encode members of the Ypt family exhibiting >80% sequence identity. Whereas the disruption of one of the two genes was phenotypically neutral, the disruption of both YPT31 and YPT32 led to lethality. Depletion of wild-type Ypt31p or of a short-lived ubiquitin-Ypt31p in a ypt32 null background led to a massive accumulation of Golgi-like membranes, an inhibition of invertase secretion and defects in vacuolar protein maturation. Similar alterations were observed in a conditional-lethal ypt31-1 mutant at 30 min after shift to the non-permissive temperature. According to subcellular fractionation, a significant part of Ypt31p appeared to be located in Golgi-enriched membrane fractions. In accordance with this, indirect immunofluorescence using affinity-purified anti-Ypt31p antibodies gave a punctate staining similar to that observed with Golgi-located proteins. From the phenotypic alterations observed in ypt31 and ypt32 mutants, it seems likely that the two GTPases are involved in intra-Golgi transport or in the formation of transport vesicles at the most distal Golgi compartment.     

Isolation of an extragenic suppressor of the rna1-1 mutation in Saccharomyces cerevisiae

The small GTPase Ran is essential for nucleocytoplasmic transport of macromolecules. In the yeast Saccharomyces cerevisiae, Rna1p functions as a Ran-GTPase activating protein (RanGAP1). Strains carrying the rna1-1 mutation exhibit defects in nuclear transport and, as a consequence, accumulate precursor tRNAs. We have isolated two recessive suppressors of the rna1-1 mutation. Further characterization of one of the suppressor mutations, srn10-1, reveals that the mutation (i) can not bypass the need for Rna1p function and (ii) suppresses the accumulation of unspliced pre-tRNA caused by rna1-1. The SRN10 gene is not essential for cell viability and encodes an acidic protein (pI = 5.27) of 24.8 kDa. Srn10p is located in the cytoplasm, as determined by indirect immunofluorescence microscopy. Two-hybrid analysis reveals that there is a physical interaction between Srn10p and Rna1p in vivo. Our results identify a protein that interacts with the yeast RanGAP1. Received: 2 March 1998 / Accepted: 17 June 1998     

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking.     

Localization of the sapA gene on a physical map of Campylobacter fetus chromosomal DNA

     We constructed a physical map of Campylobacter fetus TK(+) chromosomal DNA digested by either SmaI, SalI, or NotI using pulsed-field gel electrophoresis and Southern hybridization data. The genome size of C. fetus TK(+) is 2016 kb, larger than that reported by the others. To locate the sapA gene, which encodes the surface array protein (SAP), on the physical map, we performed Southern hybridizations with probes based on the conserved region of the sapA gene. The results showed that more than seven copies of the conserved region were present on C. fetus chromosomal DNA and that the sapA gene was located on a limited number of fragments forming a cluster of genes. By comparing fingerprint patterns of strain TK(+) and strain TK(–), which lost the ability to produce SAP during culture on agar medium, an approximately 10 kb deletion was observed in the fragments of strain TK(–). The results of Southern hybridization with two probes, one from the upstream region and the other from the variable region of sapA, suggest that the loss of SAP expression might not be the result of the loss of the sapA gene itself, but only a loss of its control systems. Received: 25 May 1994 / Accepted: 1 September 1994