© The Rockefeller University Press,
0021-9525/1997//1419 $5.00
The Journal of Cell Biology, Volume 139, Number 6,
, 1997 1419-1431
Peroxisomal Targeting, Import, and Assembly of Alcohol Oxidase in Pichia pastoris
Hans R. Waterham,
Kimberly A. Russell,
Yne de Vries, and
James M. Cregg
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000
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.
Abbreviations used in this paper: AOX, alcohol oxidase; AOX-WT, wild-type AOX; ARF, alanine-arginine-phenylalanineCOOH; CAT, catalase; FAD, flavine adenine dinucleotide; LARF, leucine-alanine-arginine-phenylalanineCOOH; MDHC, malate dehydrogenase 3; MOX; AOX from Hansenula polymorpha; MSG; methione-serine-glysine; pex, peroxisome biogenesis defective; PTS, proximal targeting signal; SKL, arginine-serine-cysteineCOOH.
This research was supported by a National Institutes of Health grant (DK-43698) and a National Science Foundation grant (MCB-9514289) to J.M. Cregg.
H.R. Waterham's present address is Department of Pediatrics, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.
Y. de Vries' present address is Department of Bacterial Genetics, Intervet B.V., P.O. Box 31, 5830 AA Boxmeer, The Netherlands.
Address all correspondence to James M. Cregg, Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, P.O. Box 91000, Portland, OR 97291-1000. Tel.: (503) 690-1217. Fax: (503) 690-1464. E-mail: cregg{at}bmb.ogi.edu

CiteULike
Complore
Connotea
Del.icio.us
Digg
Facebook
Reddit
Technorati
Twitter What's this?
This article has been cited by other articles:
-
Daniel, G., Volc, J., Filonova, L., Plihal, O., Kubatova, E., Halada, P.
(2007). Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood. Appl. Environ. Microbiol.
73: 6241-6253
[Abstract]
[Full Text]
-
Gunkel, K., van Dijk, R., Veenhuis, M., van der Klei, I. J.
(2004). Routing of Hansenula polymorpha Alcohol Oxidase: An Alternative Peroxisomal Protein-sorting Machinery. Mol. Biol. Cell
15: 1347-1355
[Abstract]
[Full Text]
-
Ozimek, P., van Dijk, R., Latchev, K., Gancedo, C., Wang, D. Y., van der Klei, I. J., Veenhuis, M.
(2003). Pyruvate Carboxylase Is an Essential Protein in the Assembly of Yeast Peroxisomal Oligomeric Alcohol Oxidase. Mol. Biol. Cell
14: 786-797
[Abstract]
[Full Text]
-
Titorenko, V. I., Nicaud, J.-M., Wang, H., Chan, H., Rachubinski, R. A.
(2002). Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica. JCB
156: 481-494
[Abstract]
[Full Text]
-
Subramani, S.
(2002). Hitchhiking fads en route to peroxisomes. JCB
156: 415-417
[Abstract]
[Full Text]
-
Horiguchi, H., Yurimoto, H., Goh, T.-K., Nakagawa, T., Kato, N., Sakai, Y.
(2001). Peroxisomal Catalase in the Methylotrophic Yeast Candida boidinii: Transport Efficiency and Metabolic Significance. J. Bacteriol.
183: 6372-6383
[Abstract]
[Full Text]
-
Stewart, M. Q., Esposito, R. D., Gowani, J., Goodman, J. M.
(2001). Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments. J. Cell Sci.
114: 2863-2868
[Abstract]
[Full Text]
-
Salomons, F. A., Kiel, J. A. K. W., Faber, K. N., Veenhuis, M., van der Klei, I. J.
(2000). Overproduction of Pex5p Stimulates Import of Alcohol Oxidase and Dihydroxyacetone Synthase in a Hansenula polymorpha pex14 Null Mutant. J. Biol. Chem.
275: 12603-12611
[Abstract]
[Full Text]
-
Ding, M, Clayton, C, Soldati, D
(2000). Toxoplasma gondii catalase: are there peroxisomes in toxoplasma?. J. Cell Sci.
113: 2409-2419
[Abstract]
-
Johnson, M. A., Waterham, H. R., Ksheminska, G. P., Fayura, L. R., Cereghino, J. L., Stasyk, O. V., Veenhuis, M., Kulachkovsky, A. R., Sibirny, A. A., Cregg, J. M.
(1999). Positive Selection of Novel Peroxisome Biogenesis-Defective Mutants of the Yeast Pichia pastoris. Genetics
151: 1379-1391
[Abstract]
[Full Text]
-
Titorenko, V. I., Smith, J. J., Szilard, R. K., Rachubinski, R. A.
(1998). Pex20p of the Yeast Yarrowia lipolytica Is Required for the Oligomerization of Thiolase in the Cytosol and for Its Targeting to the Peroxisome. JCB
142: 403-420
[Abstract]
[Full Text]
-
Titorenko, V. I., Nicaud, J.-M., Wang, H., Chan, H., Rachubinski, R. A.
(2002). Acyl-CoA oxidase is imported as a heteropentameric, cofactor-containing complex into peroxisomes of Yarrowia lipolytica. JCB
156: 481-494
[Abstract]
[Full Text]
-
Subramani, S.
(2002). Hitchhiking fads en route to peroxisomes. JCB
156: 415-417
[Abstract]
[Full Text]