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© The Rockefeller University Press, 0021-9525/1997//1419 $5.00
The Journal of Cell Biology, Volume 139, Number 6, , 1997 1419-1431

Article

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


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