scholarly journals Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

1990 ◽  
Vol 96 (4) ◽  
pp. 583-590
Author(s):  
M. Veenhuis ◽  
J.M. Goodman
Keyword(s):  
Early Stage ◽  
Morphological Changes ◽  
Polyclonal Antibodies ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Peroxisomal Membrane ◽  
Cell Induction ◽  
The Matrix ◽  
Dihydroxyacetone Synthase

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme.

1996 ◽  
Vol 134 (1) ◽  
pp. 37-51 ◽  
Author(s):  
Y Sakai ◽  
A Saiganji ◽  
H Yurimoto ◽  
K Takabe ◽  
H Saiki ◽  
...  
Keyword(s):  
Single Gene ◽  
Alcohol Oxidase ◽  
Candida Boidinii ◽  
High Electron ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
Carboxyl Terminal ◽  
Dihydroxyacetone Synthase ◽  
Physiological And Biochemical ◽  
Solute Transporters

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.

Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments

2001 ◽  
Vol 114 (15) ◽  
pp. 2863-2868
Author(s):  
Mary Q. Stewart ◽  
Renee D. Esposito ◽  
Jehangir Gowani ◽  
Joel M. Goodman
Keyword(s):  
Alcohol Oxidase ◽  
Protein Assembly ◽  
Matrix Proteins ◽  
Thiamine Pyrophosphate ◽  
Model Organisms ◽  
Candida Boidinii ◽  
Methylotrophic Yeasts ◽  
The Matrix ◽  
Cellular Compartments ◽  
Dihydroxyacetone Synthase

Alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS) constitute the bulk of matrix proteins in methylotrophic yeasts, model organisms for the study of peroxisomal assembly. Both are homooligomers; AO is a flavin-containing octamer, whereas DHAS is a thiamine pyrophosphate-containing dimer. Experiments in recent years have demonstrated that assembly of peroxisomal oligomers can occur before import; indeed the absence of chaperones within the peroxisomal matrix calls into question the ability of this compartment to assemble proteins at all. We have taken a direct pulse-chase approach to monitor import and assembly of the two major proteins of peroxisomes in Candida boidinii. Oligomers of AO are not observed in the cytosol, consistent with the proteins inability to undergo piggyback import. Indeed, oligomerization of AO can be followed within the peroxisomal matrix, directly demonstrating the capacity of this compartment for protein assembly. By contrast, DHAS quickly dimerizes in the cytosol before import. Binding and import was slowed at 15°C; the effect on AO was more dramatic. In conclusion, our data indicate that peroxisomes assemble AO in the matrix, while DHAS undergoes dimerization prior to import.

scholarly journals A Methylotrophic Pathway Participates in Pectin Utilization by Candida boidinii

2000 ◽  
Vol 66 (10) ◽  
pp. 4253-4257 ◽  
Author(s):  
Tomoyuki Nakagawa ◽  
Tatsuro Miyaji ◽  
Hiroya Yurimoto ◽  
Yasuyoshi Sakai ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Alcohol Oxidase ◽  
Pectate Lyase ◽  
Methylotrophic Yeast ◽  
Pectin Methylesterase ◽  
Candida Boidinii ◽  
Methanol Metabolism ◽  
Genes Encoding ◽  
Dihydroxyacetone Synthase ◽  
In Cells

ABSTRACT The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

scholarly journals Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins

1990 ◽  
Vol 97 (1) ◽  
pp. 193-204 ◽  
Author(s):  
J.M. Goodman ◽  
S.B. Trapp ◽  
H. Hwang ◽  
M. Veenhuis
Keyword(s):  
Oleic Acid ◽  
Membrane Proteins ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
General Function ◽  
Amino Acid Oxidase ◽  
Peroxisomal Membrane ◽  
Methanol Metabolism

Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 × 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47. Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively. This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine. the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol. In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified. Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate. The data strongly suggest that the function of PMP20 is related to methanol metabolism. In contrast, the functions of PMPs31-32 and PMP47 are ‘substrate-nonspecific’. We speculate that they may relate to the structure, assembly or general function of the organelle.

scholarly journals Preperoxisomal vesicles can form in the absence of Pex3

2014 ◽  
Vol 204 (5) ◽  
pp. 659-668 ◽  
Author(s):  
Kèvin Knoops ◽  
Selvambigai Manivannan ◽  
Małgorzata N. Cepińska ◽  
Arjen M. Krikken ◽  
Anita M. Kram ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
De Novo ◽  
Alcohol Oxidase ◽  
Matrix Proteins ◽  
Membrane Structures ◽  
Peroxisomal Membrane ◽  
Double Deletion ◽  
The Matrix ◽  
Autophagic Degradation ◽  
Mutant Cells

We demonstrate that the peroxin Pex3 is not required for the formation of peroxisomal membrane structures in yeast pex3 mutant cells. Notably, pex3 mutant cells already contain reticular and vesicular structures that harbor key proteins of the peroxisomal receptor docking complex—Pex13 and Pex14—as well as the matrix proteins Pex8 and alcohol oxidase. Other peroxisomal membrane proteins in these cells are unstable and transiently localized to the cytosol (Pex10, Pmp47) or endoplasmic reticulum (Pex11). These reticular and vesicular structures are more abundant in cells of a pex3 atg1 double deletion strain, as the absence of Pex3 may render them susceptible to autophagic degradation, which is blocked in this double mutant. Contrary to earlier suggestions, peroxisomes are not formed de novo from the endoplasmic reticulum when the PEX3 gene is reintroduced in pex3 cells. Instead, we find that reintroduced Pex3 sorts to the preperoxisomal structures in pex3 cells, after which these structures mature into normal peroxisomes.

Connection of discontinuous segments in early functional recovery from thoracic ossification of the posterior longitudinal ligament treated with posterior instrumented surgery

2020 ◽  
Vol 32 (2) ◽  
pp. 200-206
Author(s):  
Kei Ando ◽  
Kazuyoshi Kobayashi ◽  
Masaaki Machino ◽  
Kyotaro Ota ◽  
Satoshi Tanaka ◽  
...  
Keyword(s):  
Early Stage ◽  
Morphological Changes ◽  
Japanese Orthopaedic Association ◽  
Multislice Ct ◽  
Posterior Longitudinal Ligament ◽  
Ct Images ◽  
Rigid Fixation ◽  
Disc Space ◽  
Fusion Surgery ◽  
Estimated Blood Loss

OBJECTIVEThe objective of this study was to investigate the relationship between morphological changes in thoracic ossification of the posterior longitudinal ligament (T-OPLL) and postoperative neurological recovery after thoracic posterior fusion surgery. Changes of OPLL morphology and postoperative recovery in cases with T-OPLL have not been examined.METHODSIn this prospective study, the authors evaluated data from 44 patients (23 male and 21 female) who underwent posterior decompression and fusion surgery with instrumentation for the treatment of T-OPLL at our hospital. The patients’ mean age at surgery was 50.7 years (range 38–68 years). The minimum duration of follow-up was 2 years. The location of thoracic ossification of the ligamentum flavum (T-OLF), T-OLF at the OPLL level, OPLL morphology, fusion range, estimated blood loss, operative time, pre- and postoperative Japanese Orthopaedic Association (JOA) scores, and JOA recovery rate were investigated. Reconstructed sagittal multislice CT images were obtained before and at 3 and 6 months and 1 and 2 years after surgery. The basic fusion area was 3 vertebrae above and below the OPLL lesion. All parameters were compared between patients with and without continuity across the disc space at the OPLL at 3 and 6 months after surgery.RESULTSThe preoperative morphology of OPLL was discontinuous across the disc space between the rostral and caudal ossification regions on sagittal CT images in all but one of the patients. Postoperatively, these segments became continuous in 42 patients (97.7%; occurring by 6.6 months on average) without progression of OPLL thickness. Patients with continuity at 3 months had significantly lower rates of diabetes mellitus (p < 0.05) and motor palsy in the lower extremities (p < 0.01). The group with continuity also had significantly higher mean postoperative JOA scores at 3 (p < 0.01) and 6 (p < 0.05) months and mean JOA recovery rates at 3 and 6 months (both p < 0.01) after surgery.CONCLUSIONSPreoperatively, discontinuity of rostral and caudal ossified lesions was found on CT in all patients but one of this group of 44 patients who needed surgery for T-OPLL. Rigid fixation with instrumentation may have allowed these segments to connect at the OPLL. Such OPLL continuity at an early stage after surgery may accelerate spinal cord recovery.

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