Pichia pastoris Pex14p, a phosphorylated peroxisomal membrane protein, is part of a PTS-receptor docking complex and interacts with many peroxins.

The peroxisomal protein import machinery plays a central role in the assembly of this organelle in all eukaryotes. Genes encoding components of this machinery, termed peroxins or Pex proteins, have been isolated and characterized in several yeast species and in mammals, including humans. Here we report on one of these components, Pex14p, from the methylotrophic yeast Pichia pastoris. Work in other organisms has shown that Pex14p is located on the cytoplasmic surface of the peroxisomal membrane and binds peroxisomal targeting signal (PTS) receptors carrying proteins bound for the peroxisomal matrix, results that have led to the hypothesis that Pex14p is a receptor-docking protein. P. pastoris Pex14p (PpPex14p) behaves like an integral membrane protein, with its C-terminus exposed on the cytosolic side of the peroxisomal membrane. PpPex14p complexes with many peroxins, including Pex3p (Snyder et al., 1999b), Pex5p, Pex7p, Pex13p, Pex17p, itself, and a previously unreported peroxin, Pex8p. A portion of Pex14p is phosphorylated, but both phosphorylated and unphosphorylated forms of Pex14p interact with several peroxins. The interactions between Pex14p and other peroxins provide clues regarding the function of Pex14p in peroxisomal protein import.

Import of peroxisomal matrix and membrane proteins.

This review summarizes the progress made in our understanding of peroxisome biogenesis in the last few years, during which the functional roles of many of the 23 peroxins (proteins involved in peroxisomal protein import and peroxisome biogenesis) have become clearer. Previous reviews in the field have focussed on the metabolic functions of peroxisomes, aspects of import/biogenesis the role of peroxins in human disease, and involvement of the endoplasmic reticulum in peroxisome membrane biogenesis as well as the degradation of this organelle. This review refers to some of the earlier work for the sake of introduction and continuity but deals primarily with the more recent progress. The principal areas of progress are the identification of new peroxins, definition of protein-protein interactions among peroxins leading to the recognition of complexes involved in peroxisomal protein import, insight into the biogenesis of peroxisomal membrane proteins, and, of most importance, the elucidation of the role of many conserved peroxins in human disease. Given the rapid progress in the field, this review also highlights some of the unanswered questions that remain to be tackled.

The peroxin Pex19p interacts with multiple, integral membrane proteins at the peroxisomal membrane.

Pex19p is a protein required for the early stages of peroxisome biogenesis, but its precise function and site of action are unknown. We tested the interaction between Pex19p and all known Pichia pastoris Pex proteins by the yeast two-hybrid assay. Pex19p interacted with six of seven known integral peroxisomal membrane proteins (iPMPs), and these interactions were confirmed by coimmunoprecipitation. The interactions were not reduced upon inhibition of new protein synthesis, suggesting that they occur with preexisting, and not newly synthesized, pools of iPMPs. By mapping the domains in six iPMPs that interact with Pex19p and the iPMP sequences responsible for targeting to the peroxisome membrane (mPTSs), we found the majority of these sites do not overlap. Coimmunoprecipitation of Pex19p from fractions that contain peroxisomes or cytosol revealed that the interactions between predominantly cytosolic Pex19p and the iPMPs occur in the organelle pellet that contains peroxisomes. These data, taken together, suggest that Pex19p may have a chaperone-like role at the peroxisome membrane and that it is not the receptor for targeting of iPMPs to the peroxisome.

Pex17p is required for import of both peroxisome membrane and lumenal proteins and interacts with Pex19p and the peroxisome targeting signal-receptor docking complex in Pichia pastoris.

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Delta mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Delta mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal-receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.

Ulnar neuropathy as a complication of macular hole surgery.

OBJECTIVE: To report a series of patients manifesting ulnar neuropathy as an extraocular complication following macular hole surgery and facedown positioning. METHODS: Retrospective chart review of 7 patients identified by the operating surgeon as developing ulnar neuropathy during the immediate postoperative period after undergoing vitrectomy surgery with fluid-gas exchange for macular hole followed by at least 1 week of strict facedown positioning. RESULTS: All 7 patients developed symptoms of ulnar neuropathy, including paresthesias, dysesthesias, pain, weakness, and muscle atrophy. Signs included abnormal electromyogram, prolonged nerve conduction velocities, and impaired neurologic clinical test results in patients examined. Symptoms did not resolve with cessation of facedown positioning, and with follow-up ranging from 3 to 24 months all patients had persistent symptoms. All patients had positioned themselves with their arms continuously flexed. Three of 7 patients had placed pressure directly on their bent elbows. CONCLUSIONS: Ulnar neuropathy is an extraocular complication of macular hole surgery that can be attributed to arm position during postoperative facedown positioning. Surgeons performing macular hole surgery should caution their patients to minimize the amount of time spent with their elbows in a flexed position. Particular effort should be made to minimize pressure on the bent elbow.

Pex22p of Pichia pastoris, essential for peroxisomal matrix protein import, anchors the ubiquitin-conjugating enzyme, Pex4p, on the peroxisomal membrane.

We isolated a Pichia pastoris mutant that was unable to grow on the peroxisome-requiring media, methanol and oleate. Cloning the gene by complementation revealed that the encoded protein, Pex22p, is a new peroxin. A Deltapex22 strain does not grow on methanol or oleate and is unable to import peroxisomal matrix proteins. However, this strain targets peroxisomal membrane proteins to membranes, most likely peroxisomal remnants, detectable by fluorescence and electron microscopy. Pex22p, composed of 187 amino acids, is an integral peroxisomal membrane protein with its NH2 terminus in the matrix and its COOH terminus in the cytosol. It contains a 25-amino acid peroxisome membrane-targeting signal at its NH2 terminus. Pex22p interacts with the ubiquitin-conjugating enzyme Pex4p, a peripheral peroxisomal membrane protein, in vivo, and in a yeast two-hybrid experiment. Pex22p is required for the peroxisomal localization of Pex4p and in strains lacking Pex22p, the Pex4p is cytosolic and unstable. Therefore, Pex22p anchors Pex4p at the peroxisomal membrane. Strains that do not express Pex4p or Pex22p have similar phenotypes and lack Pex5p, suggesting that Pex4p and Pex22p act at the same step in peroxisome biogenesis. The Saccharomyces cerevisiae hypothetical protein, Yaf5p, is the functional homologue of P. pastoris Pex22p.

Current popularity of pneumatic retinopexy.

PURPOSE: To compare the popularity of pneumatic retinopexy (PR) in 1997 with its popularity in 1990 among retinal specialists. METHODS: In 1997, a survey was mailed to the 1994-1995 members of the Retina or Vitreous Societies who lived in the United States or Canada, asking how they would manage a hypothetical retinal detachment. The choices were limited to PR, segmental scleral buckling, scleral bucking with encircling, primary vitrectomy, and Lincoff balloon. The results of the survey were compared with those previously reported by a similar survey in 1990. RESULTS: The majority (55%) of respondents selected PR, which is a twofold increase over those who preferred it in 1990 (odds ratio 2.08; 95% confidence interval 1.53, 2.85). The popularity of PR was inversely proportional to the length of time the respondents had been in practice. If the eye with the hypothetical detachment had pseudophakia, only 30% of respondents selected PR. If the eye had additional tears, vitreous hemorrhage, or lattice degeneration, only about one-sixth preferred PR. CONCLUSION: Pneumatic retinopexy was much more popular in 1997 than it was in 1990. Its popularity continues to be influenced by the age of the surgeon and by the complexity of the detachment.

Pex19p interacts with Pex3p and Pex10p and is essential for peroxisome biogenesis in Pichia pastoris.

We report the cloning and characterization of Pichia pastoris PEX19 by complementation of a peroxisome-deficient mutant strain. Import of peroxisomal targeting signal 1- and 2-containing peroxisomal matrix proteins is defective in pex19 mutants. PEX19 encodes a hydrophilic 299-amino acid protein with sequence similarity to Saccharomyces cerevisiae Pex19p and human and Chinese hamster PxF, all farnesylated proteins, as well as hypothetical proteins from Caenorhabditis elegans and Schizosaccharomyces pombe. The farnesylation consensus is conserved in PpPex19p but dispensable for function and appears unmodified under the conditions tested. Pex19p localizes predominantly to the cytosolic fraction. Biochemical and two-hybrid analyses confirmed that Pex19p interacts with Pex3p, as seen in S. cerevisiae, but unexpectedly also with Pex10p. Two-hybrid analysis demonstrated that the amino-terminal 42 amino acids of Pex19p interact with the carboxyl-terminal 335 amino acids of Pex3p. In addition, the extreme carboxyl terminus of Pex19p (67 amino acids) is required for interaction with the amino-terminal 380 amino acids of Pex10p. Biochemical and immunofluorescence microscopy analyses of pex19Delta cells identified the membrane protein Pex3p in peroxisome remnants that were not previously observed in S. cerevisiae. These small vesicular and tubular (early) remnants are morphologically distinct from other Pppex mutant (late) remnants, suggesting that Pex19p functions at an early stage of peroxisome biogenesis.

Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component.

More than 40 vacuolar protein sorting (vps) mutants have been identified which secrete proenzyme forms of soluble vacuolar hydrolases to the cell surface. A subset of these mutants has been found to show selective defects in the sorting of two vacuolar membrane proteins. Under non-permissive conditions, vps45tsf (SEC1 homolog) and pep12/vps6tsf (endosomal t-SNARE) mutants efficiently sort alkaline phosphatase (ALP) to the vacuole while multiple soluble vacuolar proteins and the membrane protein carboxypeptidase yscS (CPS) are no longer delivered to the vacuole. Vacuolar localization of ALP in these mutants does not require transport to the plasma membrane followed by endocytic uptake, as double mutants of pep12tsf and vps45tsf with sec1 and end3 sort and mature ALP at the non-permissive temperature. Given the demonstrated role of t-SNAREs such as Pep12p in transport vesicle recognition, our results indicate that ALP and CPS are packaged into distinct transport intermediates. Consistent with ALP following an alternative route to the vacuole, isolation of a vps41tsf mutant revealed that at non-permissive temperature ALP is mislocalized while vacuolar delivery of CPS and CPY is maintained. A series of domain-swapping experiments was used to define the sorting signal that directs selective packaging and transport of ALP. Our data demonstrate that the amino-terminal 16 amino acid portion of the ALP cytoplasmic tail domain contains a vacuolar sorting signal which is responsible for the active recognition, packaging and transport of ALP from the Golgi to the vacuole via a novel delivery pathway.

Characterization of VPS41, a gene required for vacuolar trafficking and high-affinity iron transport in yeast.

Mutations in the yeast gene VPS41 give rise to poor growth on low iron medium, severe alterations in vacuolar morphology, and cause the missorting of membranous and soluble vacuolar proteins. Our studies predict that VPS41 encodes a hydrophilic protein of 992 amino acids that contains no obvious signal sequence or hydrophobic domains. The deduced Vps41p sequence contains a domain rich in glutamic and aspartic residues, as well as a domain with resemblance to a region of clathrin heavy chain. We have also identified and sequenced putative VPS41 homologues from Caenorhabditis elegans, plants, and humans. The VPS41 homologues (but not the yeast VPS41 itself) contain a conserved cysteine-rich RING-H2 zinc finger at their COOH termini. Biochemical experiments suggest that VPS41 functions in post-Golgi protein processing: the deletion mutant exhibits defective high affinity transport due to impaired Fet3p activity and also exhibits defects in the processing and sorting of multiple vacuolar hydrolases.

Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S.cerevisiae, causes proliferation of the endoplasmic reticulum membrane.

We have cloned PEX15 which is required for peroxisome biogenesis in Saccharomyces cerevisiae. pex15Delta cells are characterized by the cytosolic accumulation of peroxisomal matrix proteins containing a PTS1 or PTS2 import signal, whereas peroxisomal membrane proteins are present in peroxisomal remnants. PEX15 encodes a phosphorylated, integral peroxisomal membrane protein (Pex15p). Using multiple in vivo methods to determine the topology, Pex15p was found to be a tail-anchored type II (Ncyt-Clumen) peroxisomal membrane protein with a single transmembrane domain near its carboxy-terminus. Overexpression of Pex15p resulted in impaired peroxisome assembly, and caused profound proliferation of the endoplasmic reticulum (ER) membrane. The lumenal carboxy-terminal tail of Pex15p protrudes into the lumen of these ER membranes, as demonstrated by its O-glycosylation. Accumulation in the ER was also observed at an endogenous expression level when Pex15p was fused to the N-terminus of mature invertase. This resulted in core N-glycosylation of the hybrid protein. The lumenal C-terminal tail of Pex15p is essential for targeting to the peroxisomal membrane. Furthermore, the peroxisomal membrane targeting signal of Pex15p overlaps with an ER targeting signal on this protein. These results indicate that Pex15p may be targeted to peroxisomes via the ER, or to both organelles.

Visual field defects after macular hole surgery. A new finding.

PURPOSE: The purpose of the study is to report the problem of a temporal visual field defect occurring after macular hole surgery. METHODS: The authors reviewed the records of 13 patients found to have visual field defects after vitrectomy for macular holes. Fluorescein angiograms (13 patients), optic nerve photographs (13 patients), focal electroretinograms (3 patients), and nerve fiber analyses (8 patients) were performed in patients with visual field defects. RESULTS: An absolute, temporal, usually inferior field defect was noted in 13 patients. In eight patients, the defect was detected because of specific reports or retrospective field examination results. Five patients examined in a prospective manner were found to have field defects. No history of abnormal intraocular pressure or direct trauma to the optic nerve or retinal vessels was identified. Four patients showed optic nerve pallor and three had an anomalous-appearing disc. Focal electroretinograms were of similar amplitude in the involved retina compared to corresponding areas in the healthy fellow eye. Nerve fiber analysis showed a reduction in nerve fiber layer thickness correlating to the visual field defect in those eight patients in which this test was used. CONCLUSION: A significant temporal field defect may occur in patients after otherwise uncomplicated surgery for macular holes. The cause is unclear; however, reductions in nerve fiber layer thickness from the superior and nasal peripapillary area suggest that acute surgical release of the posterior hyaloid and the use of long-acting intraocular gas may in certain patients result in visual field defects.

Mutational activation of the Cpx signal transduction pathway of Escherichia coli suppresses the toxicity conferred by certain envelope-associated stresses.

The processing-defective outer membrane porin protein LamBA23D (Carlson and Silhavy, 1993) and a tripartite fusion protein, LamB-LacZ-PhoA (Snyder and Silhavy, 1995), are both secreted across the cytoplasmic membrane of Escherichia coli, where they exert an extracytoplasmic toxicity. Suppressors of these toxicities map to a previously characterized gene, cpxA, that encodes the sensor kinase protein of a two-component regulatory system. These activated cpxA alleles, designated as cpxA*, stimulate transcription of the periplasmic protease DegP (Danese et al., 1995), which in turn catalyses degradation of the tripartite fusion protein. In contrast, degradation of precursor LamBA23D is not significantly stimulated in a cpxA* suppressor background. In fact, increased levels of DegP in a wild-type background stabilized this protein. While a functional degP gene is required for full cpxA*-mediated suppression of both toxic envelope proteins, residual suppression is seen in cpxA* degP::Tn10 double mutants. Furthermore, cpxA* mutations suppress the toxicity conferred by the LamB-LacZ hybrid protein, which exerts its effects in the cytoplasm, sequestered from DegP. Together, these observations suggest that the activated Cpx pathway regulates additional downstream targets that contribute to suppression. A subset of these targets may constitute a regulon involved in relieving extracytoplasmic and/or secretion-related stress.

Overproduction of NlpE, a new outer membrane lipoprotein, suppresses the toxicity of periplasmic LacZ by activation of the Cpx signal transduction pathway.

The LamB-LacZ-PhoA tripartite fusion protein is secreted to the periplasm, where it exerts a toxicity of unknown origin during high-level synthesis in the presence of the inducer maltose, a phenotype referred to as maltose sensitivity. We selected multicopy suppressors of this toxicity that allow growth of the tripartite fusion strains in the presence of maltose. Mapping and subclone analysis of the suppressor locus identified a previously uncharacterized chromosomal region at 4.7 min that is responsible for suppression. DNA sequence analysis revealed a new gene with the potential to code for a protein of 236 amino acids with a predicted molecular mass of 25,829 Da. The gene product contains an amino-terminal signal sequence to direct the protein for secretion and a consensus lipoprotein modification sequence. As predicted from the sequence, the suppressor protein is labeled with [3H]palmitate and is localized to the outer membrane. Accordingly, the gene has been named nlpE (for new lipoprotein E). Increased expression of NlpE suppresses the maltose sensitivity of tripartite fusion strains and also the extracytoplasmic toxicities conferred by a mutant outer membrane protein, LamBA23D. Suppression occurs by activation of the Cpx two-component signal transduction pathway. This pathway controls the expression of the periplasmic protease DegP and other factors that can combat certain types of extracytoplasmic stress.

The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP.

DegP is a heat-shock inducible periplasmic protease in Escherichia coli. Unlike the cytoplasmic heat shock proteins, DegP is not transcriptionally regulated by the classical heat shock regulon coordinated by sigma 32. Rather, the degP gene is transcriptionally regulated by an alternate heat shock sigma factor, sigma E. Previous studies have demonstrated a signal transduction pathway that monitors the amount of outer-membrane proteins in the bacterial envelope and modulates degP levels in response to this extracytoplasmic parameter. To analyze the transcriptional regulation of degP, we examined mutations that altered transcription of a degP-lacZ operon fusion. Gain-of-function mutations in cpxA, which specifies a two-component sensor protein, stimulate transcription from degP. Defined null mutations in cpxA or the gene encoding its cognate response regulator, cpxR, decrease transcription from degP. These null mutations also prevent transcriptional induction of degP in response to overexpression of a gene specifying an envelope lipoprotein. Cpx-mediated transcription of degP is partially dependent on the activity of E sigma E, suggesting that the Cpx pathway functions in concert with E sigma E and perhaps other RNA polymerases to drive transcription of degP.

Beta-galactosidase is inactivated by intermolecular disulfide bonds and is toxic when secreted to the periplasm of Escherichia coli.

The wild-type LamB-LacZ hybrid protein inhibits the export machinery upon induction when assayed by biochemical and genetic techniques, a phenotype referred to as hybrid protein jamming. This hybrid protein also renders cells sensitive to growth in the presence of the inducer maltose, presumably because of the jamming. We constructed a new version of this fusion by adding alkaline phosphatase, encoded by phoA, to the C terminus of the LamB-LacZ hybrid protein. This tripartite protein, LamB-LacZ-PhoA, is as toxic to cells as the hybrid LamB-LacZ; however, it does not jam at temperatures greater than 33 degrees C. Extreme C-terminal sequences of LacZ function as a critical folding domain and are therefore responsible for stabilizing the LacZ structure. To determine if this region of LacZ is important for jamming, we recombined a late nonsense mutation (X90) onto the hybrid construct. We found the toxicity of this new hybrid, LamB-LacZX90, to be nearly identical to that of the full-length protein, but it also does not jam the secretion machinery. This suggests that jamming is caused by LacZ folding. We found no inhibition of secretion in the tripartite and X90 fusion strains at 37 degrees C, suggesting that the toxicity of the new fusions is novel. Under these conditions, the tripartite and X90 fusion proteins form disulfide-bonded aggregates with high molecular weights in the periplasm. Accordingly, we believe that LacZ disrupts some essential function(s) in the periplasm.

Cloning and characterization of the psaE gene of the cyanobacterium Synechococcus sp. PCC 7002: characterization of a psaE mutant and overproduction of the protein in Escherichia coli.

The psaE gene, encoding a 7.5 kDa peripheral protein of the photosystem I complex, has been cloned and characterized from the cyanobacterium Synechococcus sp. PCC 7002. The gene is transcribed as an abundant monocistronic transcript of approximately 325 nt. The PsaE protein has been overproduced in Escherichia coli, purified to homogeneity, and used to raise polyclonal antibodies. Mutant strains, in which the psaE gene was insertionally inactivated by interposon mutagenesis, were constructed and characterized. Although the PS I complexes of these strains were similar to those of the wild type, the strains grew more slowly under conditions which favour cyclic electron transport and could not grow at all under photoheterotrophic conditions. The results suggest that PsaE plays a role in cyclic electron transport in cyanobacteria.

Limiting applications of cryotherapy for severe retinopathy of prematurity.

In an effort to minimize surgical and visual morbidity of cryotherapy for retinopathy of prematurity (ROP), 18 eyes of 13 patients with 3 to 7 clock hours of stage 3 ROP with "plus" disease were treated by cryotherapy applications limited to the avascular retina adjacent to the areas of stage 3 disease. In 17 of 18 eyes, this limited use of cryotherapy was sufficient to cause regression of ROP without further treatments. After at least 3 months follow-up, ROP outcome showed a normal macular appearance in 16 eyes; two eyes developed macular dragging; no retinal detachments occurred.

Enhanced export of beta-galactosidase fusion proteins in prlF mutants is Lon dependent.

We have used fusions of the outer membrane protein LamB to beta-galactosidase (encoded by lacZ) to study the protein export process. This LamB-LacZ hybrid protein blocks export when synthesized at high levels, as evidenced by inducer (maltose) sensitivity, a phenomenon termed LacZ hybrid jamming. The prlF1 mutation relieves LacZ hybrid jamming and allows localization of the fusion protein to a noncytoplasmic compartment. prlF1 and similar alleles are gain-of-function mutations. Null mutations in this gene confer no obvious phenotypes. Extragenic suppressors of a gain-of-function prlF allele have been isolated in order to understand how this gene product affects the export process. The suppressors are all lon null mutations, and they are epistatic to all prlF phenotypes tested. Lon protease activity has been measured in prlF1 cells and shown to be increased. However, the synthesis of Lon is not increased in a prlF1 background, suggesting a previously unidentified mechanism of Lon activation. Further analysis reveals that prlF1 activates degradation of cytoplasmically localized precursors in a Lon protease-dependent manner. It is proposed that accumulation of precursors during conditions of hybrid protein jamming titrates an essential export component(s), possibly a chaperone. Increased Lon-dependent precursor degradation would free this component, thus allowing increased protein export under jamming conditions.

Pneumatic retinopexy versus scleral buckle. Preferences of Vitreous Society members, 1990.

A survey was conducted among the 1990 members of the Vitreous Society in order to measure their acceptance of pneumatic retinopexy. They were asked which treatment they would prefer should they suffer a hypothetical detachment. The choices were limited to pneumatic retinopexy or scleral buckling (encircling or segmental). The majority of respondents selected a scleral buckling procedure for a phakic retinal detachment with two adjacent superior temporal quadrant tears. Surgeons who had been in practice for 10 years or less (median for entire group = 10 years) were significantly more likely to select a pneumatic retinopexy procedure. As the details of the hypothetical detachment became more complicated with myopia, additional tears, vitreous hemorrhage, or lattice degeneration with a positive family history, the respondents selected a scleral buckling procedure with greater frequency, and the differences between the choices of the surgeons became nonsignificant. This survey shows that many surgeons feel pneumatic retinopexy is an acceptable alternative to buckling surgery in select cases. There were no trends by geographic location.