scholarly journals Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris

1997 ◽  
Vol 139 (6) ◽  
pp. 1419-1431 ◽  
Author(s):  
Hans R. Waterham ◽  
Kimberly A. Russell ◽  
Yne de Vries ◽  
James M. Cregg
Keyword(s):  
Pichia Pastoris ◽  
Matrix Protein ◽  
Alcohol Oxidase ◽  
Mutant Form ◽  
Wild Type ◽  
Epitope Tag ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Terminal Amino ◽  
Utilization Pathway

Alcohol oxidase (AOX), the first enzyme in the yeast methanol utilization pathway is a homooctameric peroxisomal matrix protein. In peroxisome biogenesis-defective (pex) mutants of the yeast Pichia pastoris, AOX fails to assemble into active octamers and instead forms inactive cytoplasmic aggregates. The apparent inability of AOX to assemble in the cytoplasm contrasts with other peroxisomal proteins that are able to oligomerize before import. To further investigate the import of AOX, we first identified its peroxisomal targeting signal (PTS). We found that sequences essential for targeting AOX are primarily located within the four COOH-terminal amino acids of the protein leucine-alanine-arginine-phenylalanine COOH (LARF). To examine whether AOX can oligomerize before import, we coexpressed AOX without its PTS along with wild-type AOX and determined whether the mutant AOX could be coimported into peroxisomes. To identify the mutant form of AOX, the COOH-terminal LARF sequence of the protein was replaced with a hemagglutinin epitope tag (AOX–HA). Coexpression of AOX–HA with wild-type AOX (AOX-WT) did not result in an increase in the proportion of AOX–HA present in octameric active AOX, suggesting that newly synthesized AOX–HA cannot oligomerize with AOX-WT in the cytoplasm. Thus, AOX cannot initiate oligomerization in the cytoplasm, but must first be targeted to the organelle before assembly begins.

scholarly journals The Hansenula polymorpha PER1 gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy- and amino-terminal targeting signals.

1994 ◽  
Vol 127 (3) ◽  
pp. 737-749 ◽  
Author(s):  
H R Waterham ◽  
V I Titorenko ◽  
P Haima ◽  
J M Cregg ◽  
W Harder ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Sequence Similarity ◽  
Hansenula Polymorpha ◽  
Matrix Proteins ◽  
Regulatory Function ◽  
Wild Type ◽  
Amino Terminal ◽  
Peroxisomal Targeting ◽  
Targeting Signal

We describe the cloning of the Hansenula polymorpha PER1 gene and the characterization of the gene and its product, PER1p. The gene was cloned by functional complementation of a per1 mutant of H. polymorpha, which was impaired in the import of peroxisomal matrix proteins (Pim- phenotype). The DNA sequence of PER1 predicts that PER1p is a polypeptide of 650 amino acids with no significant sequence similarity to other known proteins. PER1 expression was low but significant in wild-type H. polymorpha growing on glucose and increased during growth on any one of a number of substrates which induce peroxisome proliferation. PER1p contains both a carboxy- (PTS1) and an amino-terminal (PTS2) peroxisomal targeting signal which both were demonstrated to be capable of directing bacterial beta-lactamase to the organelle. In wild-type H. polymorpha PER1p is a protein of low abundance which was demonstrated to be localized in the peroxisomal matrix. Our results suggest that the import of PER1p into peroxisomes is a prerequisite for the import of additional matrix proteins and we suggest a regulatory function of PER1p on peroxisomal protein support.

scholarly journals Transport of microinjected alcohol oxidase from Pichia pastoris into vesicles in mammalian cells: involvement of the peroxisomal targeting signal.

1992 ◽  
Vol 118 (3) ◽  
pp. 499-508 ◽  
Author(s):  
P A Walton ◽  
S J Gould ◽  
R A Rachubinski ◽  
S Subramani ◽  
J R Feramisco
Keyword(s):  
Pichia Pastoris ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Protein Transport ◽  
Zellweger Syndrome ◽  
Alcohol Oxidase ◽  
Peroxisomal Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

This report describes the microinjection of a purified peroxisomal protein, alcohol oxidase, from Pichia pastoris into mammalian tissue culture cells and the subsequent transport of this protein into vesicular structures. Transport was into membrane-enclosed vesicles as judged by digitonin-permeabilization experiments. The transport was time and temperature dependent. Vesicles containing alcohol oxidase could be detected as long as 6 d after injection. Coinjection of synthetic peptides containing a consensus carboxyterminal tripeptide peroxisomal targeting signal resulted in abolition of alcohol oxidase transport into vesicles in all cell lines examined. Double-label experiments indicated that, although some of the alcohol oxidase was transported into vesicles that contained other peroxisomal proteins, the bulk of the alcohol oxidase did not appear to be transported to preexisting peroxisomes. While the inhibition of transport of alcohol oxidase by peptides containing the peroxisomal targeting signal suggests a competition for some limiting component of the machinery involved in the sorting of proteins into peroxisomes, the organelles into which the majority of the protein is targeted appear to be unusual and distinct from endogenous peroxisomes by several criteria. Microinjected alcohol oxidase was transported into vesicles in normal fibroblasts and also in cell lines derived from patients with Zellweger syndrome, which are unable to transport proteins containing the ser-lys-leu-COOH peroxisomal targeting signal into peroxisomes (Walton et al., 1992). The implications of this result for the mechanism of peroxisomal protein transport are discussed.

scholarly journals Enlarged Peroxisomes Are Present in Oleic Acid–grown Yarrowia lipolytica Overexpressing the PEX16 Gene Encoding an Intraperoxisomal Peripheral Membrane Peroxin

1997 ◽  
Vol 137 (6) ◽  
pp. 1265-1278 ◽  
Author(s):  
Gary A. Eitzen ◽  
Rachel K. Szilard ◽  
Richard A. Rachubinski
Keyword(s):  
Oleic Acid ◽  
Yarrowia Lipolytica ◽  
Consensus Sequence ◽  
Wild Type ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
Peripheral Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal

Pex mutants of the yeast Yarrowia lipolytica are defective in peroxisome assembly. The mutant strain pex16-1 lacks morphologically recognizable peroxisomes. Most peroxisomal proteins are mislocalized to a subcellular fraction enriched for cytosol in pex16 strains, but a subset of peroxisomal proteins is localized at, or near, wild-type levels to a fraction typically enriched for peroxisomes. The PEX16 gene was isolated by functional complementation of the pex16-1 strain and encodes a protein, Pex16p, of 391 amino acids (44,479 D). Pex16p has no known homologues. Pex16p is a peripheral protein located at the matrix face of the peroxisomal membrane. Substitution of the carboxylterminal tripeptide Ser-Thr-Leu, which is similar to the consensus sequence of peroxisomal targeting signal 1, does not affect targeting of Pex16p to peroxisomes. Pex16p is synthesized in wild-type cells grown in glucose-containing media, and its levels are modestly increased by growth of cells in oleic acid–containing medium. Overexpression of the PEX16 gene in oleic acid– grown Y. lipolytica leads to the appearance of a small number of enlarged peroxisomes, which contain the normal complement of peroxisomal proteins at levels approaching those of wild-type peroxisomes.

Metabolic control of peroxisome abundance

1999 ◽  
Vol 112 (10) ◽  
pp. 1579-1590 ◽  
Author(s):  
C.C. Chang ◽  
S. South ◽  
D. Warren ◽  
J. Jones ◽  
A.B. Moser ◽  
...  
Keyword(s):  
Metabolic Control ◽  
Matrix Protein ◽  
Protein Import ◽  
Zellweger Syndrome ◽  
Mutant Gene ◽  
Peroxisomal Membrane ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Peroxisomal Targeting Signal

Zellweger syndrome and related disorders represent a group of lethal, genetically heterogeneous diseases. These peroxisome biogenesis disorders (PBDs) are characterized by defective peroxisomal matrix protein import and comprise at least 10 complementation groups. The genes defective in seven of these groups and more than 90% of PBD patients are now known. Here we examine the distribution of peroxisomal membrane proteins in fibroblasts from PBD patients representing the seven complementation groups for which the mutant gene is known. Peroxisomes were detected in all PBD cells, indicating that the ability to form a minimal peroxisomal structure is not blocked in these mutants. We also observed that peroxisome abundance was reduced fivefold in PBD cells that are defective in the PEX1, PEX5, PEX12, PEX6, PEX10, and PEX2 genes. These cell lines all display a defect in the import of proteins with the type-1 peroxisomal targeting signal (PTS1). In contrast, peroxisome abundance was unaffected in cells that are mutated in PEX7 and are defective only in the import of proteins with the type-2 peroxisomal targeting signal. Interestingly, a fivefold reduction in peroxisome abundance was also observed for cells lacking either of two PTS1-targeted peroxisomal beta-oxidation enzymes, acyl-CoA oxidase and 2-enoyl-CoA hydratase/D-3-hydroxyacyl-CoA dehydrogenase. These results indicate that reduced peroxisome abundance in PBD cells may be caused by their inability to import these PTS1-containing enzymes. Furthermore, the fact that peroxisome abundance is influenced by peroxisomal 105-oxidation activities suggests that there may be metabolic control of peroxisome abundance.

scholarly journals The unique degradation pathway of the PTS2 receptor, Pex7, is dependent on the PTS receptor/coreceptor, Pex5 and Pex20

2014 ◽  
Vol 25 (17) ◽  
pp. 2634-2643 ◽  
Author(s):  
Danielle Hagstrom ◽  
Changle Ma ◽  
Soumi Guha-Polley ◽  
Suresh Subramani
Keyword(s):  
Matrix Protein ◽  
Protein Import ◽  
Degradation Pathway ◽  
Matrix Proteins ◽  
Receptor Recycling ◽  
Wild Type ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signals ◽  
The Stability

Peroxisomal matrix protein import uses two peroxisomal targeting signals (PTSs). Most matrix proteins use the PTS1 pathway and its cargo receptor, Pex5. The PTS2 pathway is dependent on another receptor, Pex7, and its coreceptor, Pex20. We found that during the matrix protein import cycle, the stability and dynamics of Pex7 differ from those of Pex5 and Pex20. In Pichia pastoris, unlike Pex5 and Pex20, Pex7 is constitutively degraded in wild-type cells but is stabilized in pex mutants affecting matrix protein import. Degradation of Pex7 is more prevalent in cells grown in methanol, in which the PTS2 pathway is nonessential, in comparison with oleate, suggesting regulation of Pex7 turnover. Pex7 must shuttle into and out of peroxisomes before it is polyubiquitinated and degraded by the proteasome. The shuttling of Pex7, and consequently its degradation, is dependent on the receptor recycling pathways of Pex5 and Pex20 and relies on an interaction between Pex7 and Pex20. We also found that blocking the export of Pex20 from peroxisomes inhibits PTS1-mediated import, suggesting sharing of limited components in the export of PTS receptors/coreceptors. The shuttling and stability of Pex7 are divergent from those of Pex5 and Pex20, exemplifying a novel interdependence of the PTS1 and PTS2 pathways.

scholarly journals Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome

1998 ◽  
Vol 142 (2) ◽  
pp. 403-420 ◽  
Author(s):  
Vladimir I. Titorenko ◽  
Jennifer J. Smith ◽  
Rachel K. Szilard ◽  
Richard A. Rachubinski
Keyword(s):  
Yarrowia Lipolytica ◽  
High Speed ◽  
Matrix Protein ◽  
Peroxisomal Membrane ◽  
Amino Terminal ◽  
Peroxisomal Targeting ◽  
In The Wild ◽  
Targeting Signal ◽  
Polypeptide Chains ◽  
Yeast Yarrowia Lipolytica

Pex mutants are defective in peroxisome assembly. In the pex20-1 mutant strain of the yeast Yarrowia lipolytica, the peroxisomal matrix protein thiolase is mislocalized exclusively to the cytosol, whereas the import of other peroxisomal proteins is unaffected. The PEX20 gene was isolated by functional complementation of the pex20-1 strain and encodes a protein, Pex20p, of 424 amino acids (47,274 D). Despite its role in the peroxisomal import of thiolase, which is targeted by an amino-terminal peroxisomal targeting signal-2 (PTS2), Pex20p does not exhibit homology to Pex7p, which acts as the PTS2 receptor. Pex20p is mostly cytosolic, whereas 4–8% is associated with high-speed (200,000 g) pelletable peroxisomes. In the wild-type strain, all newly synthesized thiolase is associated with Pex20p in a heterotetrameric complex composed of two polypeptide chains of each protein. This association is independent of PTS2. Pex20p is required for both the oligomerization of thiolase in the cytosol and its targeting to the peroxisome. Our data suggest that monomeric Pex20p binds newly synthesized monomeric thiolase in the cytosol and promotes the formation of a heterotetrameric complex of these two proteins, which could further bind to the peroxisomal membrane. Translocation of the thiolase homodimer into the peroxisomal matrix would release Pex20p monomers back to the cytosol, thereby permitting a new cycle of binding-oligomerization-targeting-release for Pex20p and thiolase.

scholarly journals Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.

1994 ◽  
Vol 5 (8) ◽  
pp. 829-837 ◽  
Author(s):  
M E Evers ◽  
V I Titorenko ◽  
I J van der Klei ◽  
W Harder ◽  
M Veenhuis
Keyword(s):  
Molecular Chaperone ◽  
Matrix Protein ◽  
Protein Import ◽  
Flavin Adenine Dinucleotide ◽  
Hansenula Polymorpha ◽  
Alcohol Oxidase ◽  
Auxotrophic Mutant ◽  
Matrix Proteins ◽  
Wild Type ◽  
Wild Type Control

The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented.

scholarly journals Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

1998 ◽  
Vol 141 (3) ◽  
pp. 625-636 ◽  
Author(s):  
Yasuyoshi Sakai ◽  
Antonius Koller ◽  
Linda K. Rangell ◽  
Gilbert A. Keller ◽  
Suresh Subramani
Keyword(s):  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Pyridinium Dibromide ◽  
Shed Light

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

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