I am an emeritus Professor of Microbiology, expert in antibiotic mechanisms and resistance, with a broad range of research interests in yeast cell biology, yeast kiler viruses, membrane proteins and prions.
Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from pe... more Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from peptone-and succinate-induced rod stage cells. Undegraded polysaccharide backbones of the peptidoglycans were isolated from myxobacter AL-1 protease digests by ECTEOLA cellulose and Sephadex G-50 chromatography. The polysaccharide backbones of the sphere cell wall peptidoglycan are heterogeneous in their size, and average less than 40 hexosamines per chain. Those of the rod cell walls are homogeneous in size and average 114 to 135 hexosamines per chain.
The mechanism of solubilization of the cell walls of Staphylococcus aureus by the LII enzyme from... more The mechanism of solubilization of the cell walls of Staphylococcus aureus by the LII enzyme from Flavobacterium has been investigated. This enzyme is an endopeptidase which catalyzes the hydrolysis of both D-alanyl-glycine and glycyl-glycine linkages (in the ratio 3 to 7) in the interpeptide bridge which interconnects peptidoglycan strands in the cell wall of S. ailreus strain Copenhagen. In intact cell walls of this strain
At laparotomy on infants, liver biopsy specimens weighing cu. I g (wet weight) may be obtained wi... more At laparotomy on infants, liver biopsy specimens weighing cu. I g (wet weight) may be obtained with little risk. A general method of fractionation has been used for analysis of the polysaccharides present in this amount of tissue and should prove useful for those conditions involving some metabolic disturbance (e.g. gargoylism, glycogen storage disease, amyloidosis, etc.) where abnormal storage of a polysaccharide constituent occurs. The fractionation has been applied to liver biopsy specimens from two infants, both of whom had grossly enlarged livers, in order to determine whether they had glycogen storage disease. Glycogen storage diseases result from disturbances in the enzymic degradation of glycogen in the liver due to deficiencies in either glucose-6-phosphatase or amylo-1, 6-glucosidase'. ILLINGWORTH AND CORI 2 found that glycogens in eight out of ten cases of glycogen storage disease had a normal structure, while one had abnormally short outer chains presumably due to amylo-r, 6-glucosidase deficiency. Under controlled conditions 430,ug of phosphorus was liberated from glucose-6-phosphate by IOO mg of normal liver, while IOO mg of liver from cases of glycogen storage disease liberated 20, 20, 150, 2So and 4oopg respectively. IOO mg of the liver deficient in amylo-r, 6-glucosidase liberated ISO pg.
A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus s... more A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spo VA or spoIVA (spoIIA +) strains, subclones of pRC 12, lacking a functional spoIIA gene, did complement these mutations. pRC 12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.
VOL 184 filtrate with MnO 2 yields an apparent retention of 80 per cent instead of the value of 8... more VOL 184 filtrate with MnO 2 yields an apparent retention of 80 per cent instead of the value of 8 per cent which is observed if the first Mn0 2 treatment is omitted. The same result is obtained following the addition of Mn 2 0 3 or Mn(Cl0,) 2 • In the absence of water these substances thus cause the conversion of most of the active species to a form which is not made removable by subsequent addition of water and shaking with MnO 2 • The high apparent retention of 80 per cent is not due to conversion of the active species to permanganate since the retention as permanganate, determined after dilution and precipitation of silver permanganate, is 8 per cent. The increase in permanganate retention which was observed when the diluted solution was treated with barium hydroxide suggests that the remaining 72 per cent of the activity was present as colloidal Mn0 2 , not easily removable by shaking with solid Mn0 2 • In conclusion, it has not been demonstrated that the active species in dry pyridine solution is similar in its reactions to the active species in the solid. After addition of water to the pyridine solution, the active species appears to be present as manganese dioxide which, under some conditions, forms a colloidal dispersion in the pyridine-water solution. We are grateful to the National Research Council of Canada for generous financial assistance.
A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable o... more A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable of lysing cells of Staphylococcus aureus was purified approximately 500-fold by passage through diethylaminoethyl cellulose and chromatography on carboxymethyl-cellulose. The purified enzyme was shown to act as an endopeptidase, cleaving the pentaglycine cross-bridges of the cell wall peptidoglycan at D-alanyl-glycine and glycyl-glycine linkages with the release of di-, tri-, and tetraglycine fragments. Release of NH2-alanine indicated weak N-acetylmuramyl-L-ala-' Journal paper no. J-5972 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1384.
Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer... more Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecularweight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.
nteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to pr... more nteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH 2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were E expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH 2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.
Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by trea... more Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis, an N-acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C-and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one L-alanine residue. In sphere cell walls, L-alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C-and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall. 'H ET AL.; J. BACTERIOL. Minn.). Water was used for equilibration and elution. All columns were run at room temperature. RESULTS N-and C-terminal amino acids in whole cell
SummaryThe K1 killer toxin of Saccharomyces cerevisiae consists of 103‐ and 83‐residue α and β co... more SummaryThe K1 killer toxin of Saccharomyces cerevisiae consists of 103‐ and 83‐residue α and β components whose derivation, from a 316‐residue precursor preprotoxin, requires processing at the αN‐terminus (after ProArg‐44), the αC‐terminus (after ArgArg‐149) and at the βN‐terminus (after LysArg–233). These processing events occur after translocation to the Golgi and have been investigated using β‐lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala‐26, was confirmed by N‐terminal sequence analysis of Ala‐34 and IIe‐52 fusions. Cleavage at all of the other predicted processing sites, including ProArg‐44, is dependent on activity of the Kex2 protease. A fourth Kex2‐dependent cleavage occurs at LysArg‐188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.
Frontiers in Cell and Developmental Biology, Aug 3, 2023
DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but sti... more DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but still high fidelity. In contrast, for the estimated 33% of the human proteome that is inserted as transmembrane (TM) proteins, insertion with a non-functional inverted topology is frequent. Correct topology is essential for function and trafficking to appropriate cellular compartments and is controlled principally by responses to charged residues within 15 residues of the inserted TM domain (TMD); the flank with the higher positive charge remains in the cytosol (inside), following the positive inside rule (PIR). Yeast (Saccharomyces cerevisiae) mutants that increase insertion contrary to the PIR were selected. Mutants with strong phenotypes were found only in SPF1 and STE24 (human cell orthologs are ATP13A1 and ZMPSte24) with, at the time, no known relevant functions. Spf1/Atp13A1 is now known to dislocate to the cytosol TM proteins inserted contrary to the PIR, allowing energy-conserving reinsertion. We hypothesize that Spf1 and Ste24 both recognize the short, positively charged ER luminal peptides of TM proteins inserted contrary to the PIR, accepting these peptides into their large membrane-spanning, water-filled cavities through interaction with their many interior surface negative charges. While entry was demonstrated for Spf1, no published evidence directly demonstrates substrate entry to the Ste24 cavity, internal access to its zinc metalloprotease (ZMP) site, or active withdrawal of fragments, which may be essential for function. Spf1 and Ste24 comprise a PIR quality control system that is conserved in all eukaryotes and presumably evolved in prokaryotic progenitors as they gained differentiated membrane functions. About 75% of the PIR is imposed by this quality control system, which joins the UPR, ERAD, and autophagy (ER-phagy) in coordinated, overlapping quality control of ER protein function.
Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec... more Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (secl). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In secl8 the protoxin was stable after a chase; but in sec7 and seci the protoxin was unstable, and in secl 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-L-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and secl cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-secl8 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kexi but unstable in kex2. Protoxin stability in kexi kex2 double mutants indicated the order kexi-* kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEXI-and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion. Type 1 killer Saccharomyces cerevisiae cells contain Ml double-stranded RNA (dsRNA), a
Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae a-facto... more Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae a-factor, and lIa, the secreted form of P-lactamase encoded by the bla gene of pBR322. The Ste2 and fla components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in ,la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was ceUl associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of DIa; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in Pla secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of (la activity occurred, which is consistent with inversion of the orientation of the Pla reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced (la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of (la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.
The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian micr... more The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the eR translocon and substrates in transit. Most inserted TM1 has its N-terminus in the eR lumen (Ntm orientation), as expected for arrest of normal secretion. however, 20% is found in inverted ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between ctmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a c-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. however, secretion of PrP by the mammalian translocon requires the TRaP complex, absent in yeast, where essentially all PrP ends up as TM species, 85-90% Ntm and 10-15% ctm. although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and ctm orientation. an increase in Δch from-5 to-7 caused a marked increase in ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.
Summary SCG1/GPA1, STE4, and STE18 encode the α, β and λ components of the G protein involved in ... more Summary SCG1/GPA1, STE4, and STE18 encode the α, β and λ components of the G protein involved in mating pheromone signal transduction in Saccharomyces cerevisiae. Responses, including G1 arrest and expression of genes such as FUS1, are activated by βλ, which is negatively controlled by α(GDP), We previously demonstrated that overexpression of Scg1 suppresses responses to α factor and that expression of certain hybrids between Scg1 and mammalian Gα proteins has the same effect and also suppresses growth arrest in an scg1‐null mutant. Effects were attributed to sequestration of βλ. We now show that effects on growth rate, morphology and FUS1 expression are consistent with this model. The STE4HPL allele causes dominant activation of the response pathway, and is presumed to encode a β subunit insensitive to control by α(GDP). Scg1 overexpression suppresses the growth arrest due to STE4HPL; normal α‐factor responses and fertility are restored. A model based on sequestration of βγ reconciles this result with the apparent paradox that the same level of Scg1 overexpression inhibits responses and mating in wild‐type cells. A Gαi hybrid also restores growth and allows inefficient mating in the STEHPL strain.
Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from pe... more Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from peptone-and succinate-induced rod stage cells. Undegraded polysaccharide backbones of the peptidoglycans were isolated from myxobacter AL-1 protease digests by ECTEOLA cellulose and Sephadex G-50 chromatography. The polysaccharide backbones of the sphere cell wall peptidoglycan are heterogeneous in their size, and average less than 40 hexosamines per chain. Those of the rod cell walls are homogeneous in size and average 114 to 135 hexosamines per chain.
The mechanism of solubilization of the cell walls of Staphylococcus aureus by the LII enzyme from... more The mechanism of solubilization of the cell walls of Staphylococcus aureus by the LII enzyme from Flavobacterium has been investigated. This enzyme is an endopeptidase which catalyzes the hydrolysis of both D-alanyl-glycine and glycyl-glycine linkages (in the ratio 3 to 7) in the interpeptide bridge which interconnects peptidoglycan strands in the cell wall of S. ailreus strain Copenhagen. In intact cell walls of this strain
At laparotomy on infants, liver biopsy specimens weighing cu. I g (wet weight) may be obtained wi... more At laparotomy on infants, liver biopsy specimens weighing cu. I g (wet weight) may be obtained with little risk. A general method of fractionation has been used for analysis of the polysaccharides present in this amount of tissue and should prove useful for those conditions involving some metabolic disturbance (e.g. gargoylism, glycogen storage disease, amyloidosis, etc.) where abnormal storage of a polysaccharide constituent occurs. The fractionation has been applied to liver biopsy specimens from two infants, both of whom had grossly enlarged livers, in order to determine whether they had glycogen storage disease. Glycogen storage diseases result from disturbances in the enzymic degradation of glycogen in the liver due to deficiencies in either glucose-6-phosphatase or amylo-1, 6-glucosidase'. ILLINGWORTH AND CORI 2 found that glycogens in eight out of ten cases of glycogen storage disease had a normal structure, while one had abnormally short outer chains presumably due to amylo-r, 6-glucosidase deficiency. Under controlled conditions 430,ug of phosphorus was liberated from glucose-6-phosphate by IOO mg of normal liver, while IOO mg of liver from cases of glycogen storage disease liberated 20, 20, 150, 2So and 4oopg respectively. IOO mg of the liver deficient in amylo-r, 6-glucosidase liberated ISO pg.
A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus s... more A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spo VA or spoIVA (spoIIA +) strains, subclones of pRC 12, lacking a functional spoIIA gene, did complement these mutations. pRC 12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.
VOL 184 filtrate with MnO 2 yields an apparent retention of 80 per cent instead of the value of 8... more VOL 184 filtrate with MnO 2 yields an apparent retention of 80 per cent instead of the value of 8 per cent which is observed if the first Mn0 2 treatment is omitted. The same result is obtained following the addition of Mn 2 0 3 or Mn(Cl0,) 2 • In the absence of water these substances thus cause the conversion of most of the active species to a form which is not made removable by subsequent addition of water and shaking with MnO 2 • The high apparent retention of 80 per cent is not due to conversion of the active species to permanganate since the retention as permanganate, determined after dilution and precipitation of silver permanganate, is 8 per cent. The increase in permanganate retention which was observed when the diluted solution was treated with barium hydroxide suggests that the remaining 72 per cent of the activity was present as colloidal Mn0 2 , not easily removable by shaking with solid Mn0 2 • In conclusion, it has not been demonstrated that the active species in dry pyridine solution is similar in its reactions to the active species in the solid. After addition of water to the pyridine solution, the active species appears to be present as manganese dioxide which, under some conditions, forms a colloidal dispersion in the pyridine-water solution. We are grateful to the National Research Council of Canada for generous financial assistance.
A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable o... more A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable of lysing cells of Staphylococcus aureus was purified approximately 500-fold by passage through diethylaminoethyl cellulose and chromatography on carboxymethyl-cellulose. The purified enzyme was shown to act as an endopeptidase, cleaving the pentaglycine cross-bridges of the cell wall peptidoglycan at D-alanyl-glycine and glycyl-glycine linkages with the release of di-, tri-, and tetraglycine fragments. Release of NH2-alanine indicated weak N-acetylmuramyl-L-ala-' Journal paper no. J-5972 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 1384.
Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer... more Extensively washed, dormant spores of Bacillus subtilis were disrupted with glass beads in buffer at pH 7 in the presence of protease inhibitors. Approximately 31% of the total spore protein was soluble, and another 14% was removed from the insoluble fraction by hydrolysis with lysozyme and washing with 1 M KCl and 0.1% sodium dodecyl sulfate. The residual spore integuments comprised 55% of the total spore proteins and consisted of coats and residual membrane components. Treatment of integuments with sodium dodecyl sulfate and reducing agents at pH 10 solubilized 40% of the total spore protein. Seven low-molecularweight polypeptide components of this solubilized fraction comprised 27% of the total spore protein. They are not normal membrane components and reassociated to form fibrillar structures resembling spore coat fragments. The residual insoluble material (15% of the total spore protein) was rich in cysteine and was probably also derived from the spore coats. A solubilized coat polypeptide of molecular weight 12,200 has been purified in good yield (4 to 5% of the total spore protein). Five amino acids account for 92% of its total amino acid residues: glycine, 19%; tyrosine, 31%; proline, 23%; arginine, 13%; and phenylalanine, 6%.
nteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to pr... more nteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH 2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were E expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH 2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.
Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by trea... more Cell walls of Arthrobacter crystallopoietes grown as spheres and as rods were solubilized by treatment with the B enzyme from Chalaropsis, an N-acetylmuramidase. The neutral glycopeptides were then isolated by chromatography on ECTEOLA cellulose. The glycopeptides, consisting of disaccharide-peptide units interlinked by peptide cross-bridges, were fractionated by gel filtration on Sephadex columns into oligomers of various sizes. The size distribution ranged from monomers with no cross-bridges to polymers with a high degree of polymerization, but did not differ significantly between cell walls from cells grown as spheres or rods. Some small differences in the distribution of C-and N-terminal amino acids were found. Analyses revealed that all the peptide bridges in the glycopeptide fractions from rod cell walls were formed by one L-alanine residue. In sphere cell walls, L-alanine was also found, but, in addition, higher oligomers of the glycopeptide contained glycine in their cross-bridges. These results were confirmed by determinations of C-and N-terminal amino acids released after lysostaphin and AL-1 enzyme digestions and by Edman degradations. Models representing the structures of the sphere and rod cell walls are presented. These structures indicate that the sphere cell wall is probably a more loosely knit macromolecule than is the rod cell wall. 'H ET AL.; J. BACTERIOL. Minn.). Water was used for equilibration and elution. All columns were run at room temperature. RESULTS N-and C-terminal amino acids in whole cell
SummaryThe K1 killer toxin of Saccharomyces cerevisiae consists of 103‐ and 83‐residue α and β co... more SummaryThe K1 killer toxin of Saccharomyces cerevisiae consists of 103‐ and 83‐residue α and β components whose derivation, from a 316‐residue precursor preprotoxin, requires processing at the αN‐terminus (after ProArg‐44), the αC‐terminus (after ArgArg‐149) and at the βN‐terminus (after LysArg–233). These processing events occur after translocation to the Golgi and have been investigated using β‐lactamase fusions. Signal peptidase cleavage of the precursor, predicted to occur after Ala‐26, was confirmed by N‐terminal sequence analysis of Ala‐34 and IIe‐52 fusions. Cleavage at all of the other predicted processing sites, including ProArg‐44, is dependent on activity of the Kex2 protease. A fourth Kex2‐dependent cleavage occurs at LysArg‐188. Implications for the specificity of Kex2 cleavage and preprotoxin processing are discussed.
Frontiers in Cell and Developmental Biology, Aug 3, 2023
DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but sti... more DNA replication, transcription, and translation in eukaryotic cells occur with decreasing but still high fidelity. In contrast, for the estimated 33% of the human proteome that is inserted as transmembrane (TM) proteins, insertion with a non-functional inverted topology is frequent. Correct topology is essential for function and trafficking to appropriate cellular compartments and is controlled principally by responses to charged residues within 15 residues of the inserted TM domain (TMD); the flank with the higher positive charge remains in the cytosol (inside), following the positive inside rule (PIR). Yeast (Saccharomyces cerevisiae) mutants that increase insertion contrary to the PIR were selected. Mutants with strong phenotypes were found only in SPF1 and STE24 (human cell orthologs are ATP13A1 and ZMPSte24) with, at the time, no known relevant functions. Spf1/Atp13A1 is now known to dislocate to the cytosol TM proteins inserted contrary to the PIR, allowing energy-conserving reinsertion. We hypothesize that Spf1 and Ste24 both recognize the short, positively charged ER luminal peptides of TM proteins inserted contrary to the PIR, accepting these peptides into their large membrane-spanning, water-filled cavities through interaction with their many interior surface negative charges. While entry was demonstrated for Spf1, no published evidence directly demonstrates substrate entry to the Ste24 cavity, internal access to its zinc metalloprotease (ZMP) site, or active withdrawal of fragments, which may be essential for function. Spf1 and Ste24 comprise a PIR quality control system that is conserved in all eukaryotes and presumably evolved in prokaryotic progenitors as they gained differentiated membrane functions. About 75% of the PIR is imposed by this quality control system, which joins the UPR, ERAD, and autophagy (ER-phagy) in coordinated, overlapping quality control of ER protein function.
Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec... more Killer toxin secretion was blocked at the restrictive temperature in Saccharomyces cerevisiae sec mutants with conditional defects in the S. cerevisiae secretory pathway leading to accumulation of endoplasmic reticulum (sec18), Golgi (sec7), or secretory vesicles (secl). A 43,000-molecular-weight (43K) glycosylated protoxin was found by pulse-labeling in all sec mutants at the restrictive temperature. In secl8 the protoxin was stable after a chase; but in sec7 and seci the protoxin was unstable, and in secl 11K toxin was detected in cell lysates. The chymotrypsin inhibitor tosyl-L-phenylalanyl chloromethyl ketone (TPCK) blocked toxin secretion in vivo in wild-type cells by inhibiting protoxin cleavage. The unstable protoxin in wild-type and in sec7 and secl cells at the restrictive temperature was stabilized by TPCK, suggesting that the protoxin cleavage was post-secl8 and was mediated by a TPCK-inhibitable protease. Protoxin glycosylation was inhibited by tunicamycin, and a 36K protoxin was detected in inhibited cells. This 36K protoxin was processed, but toxin secretion was reduced 10-fold. We examined two kex mutants defective in toxin secretion; both synthesized a 43K protoxin, which was stable in kexi but unstable in kex2. Protoxin stability in kexi kex2 double mutants indicated the order kexi-* kex2 in the protoxin processing pathway. TPCK did not block protoxin instability in kex2 mutants. This suggested that the KEXI-and KEX2-dependent steps preceded the sec7 Golgi block. We attempted to localize the protoxin in S. cerevisiae cells. Use of an in vitro rabbit reticulocyte-dog pancreas microsomal membrane system indicated that protoxin synthesized in vitro could be inserted into and glycosylated by the microsomal membranes. This membrane-associated protoxin was protected from trypsin proteolysis. Pulse-chased cells or spheroplasts, with or without TPCK, failed to secrete protoxin. The protoxin may not be secreted into the lumen of the endoplasmic reticulum, but may remain membrane associated and may require endoproteolytic cleavage for toxin secretion. Type 1 killer Saccharomyces cerevisiae cells contain Ml double-stranded RNA (dsRNA), a
Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae a-facto... more Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae a-factor, and lIa, the secreted form of P-lactamase encoded by the bla gene of pBR322. The Ste2 and fla components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in ,la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was ceUl associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of DIa; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in Pla secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of (la activity occurred, which is consistent with inversion of the orientation of the Pla reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced (la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of (la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.
The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian micr... more The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the eR translocon and substrates in transit. Most inserted TM1 has its N-terminus in the eR lumen (Ntm orientation), as expected for arrest of normal secretion. however, 20% is found in inverted ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between ctmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a c-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. however, secretion of PrP by the mammalian translocon requires the TRaP complex, absent in yeast, where essentially all PrP ends up as TM species, 85-90% Ntm and 10-15% ctm. although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and ctm orientation. an increase in Δch from-5 to-7 caused a marked increase in ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.
Summary SCG1/GPA1, STE4, and STE18 encode the α, β and λ components of the G protein involved in ... more Summary SCG1/GPA1, STE4, and STE18 encode the α, β and λ components of the G protein involved in mating pheromone signal transduction in Saccharomyces cerevisiae. Responses, including G1 arrest and expression of genes such as FUS1, are activated by βλ, which is negatively controlled by α(GDP), We previously demonstrated that overexpression of Scg1 suppresses responses to α factor and that expression of certain hybrids between Scg1 and mammalian Gα proteins has the same effect and also suppresses growth arrest in an scg1‐null mutant. Effects were attributed to sequestration of βλ. We now show that effects on growth rate, morphology and FUS1 expression are consistent with this model. The STE4HPL allele causes dominant activation of the response pathway, and is presumed to encode a β subunit insensitive to control by α(GDP). Scg1 overexpression suppresses the growth arrest due to STE4HPL; normal α‐factor responses and fertility are restored. A model based on sequestration of βγ reconciles this result with the apparent paradox that the same level of Scg1 overexpression inhibits responses and mating in wild‐type cells. A Gαi hybrid also restores growth and allows inefficient mating in the STEHPL strain.
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Papers by Donald Tipper