© The Rockefeller University Press,
0021-9525/1998//625 $5.00
The Journal of Cell Biology, Volume 141, Number 3,
, 1998 625-636
Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates
Yasuyoshi Sakai*,
Antonius Koller*,
Linda K. Rangell
,
Gilbert A. Keller
, and
Suresh Subramani*
* Department of Biology, University of California, San Diego, La Jolla, California 92093-0322; and
Pharmacological Science, Genentech, South San Francisco, California 94080
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.
Abbreviations used in this paper: ABTS, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid); AOX, alcohol oxidase; DHAS, dihydroxyacetone synthase; FM4-64, N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide; GFP, green fluorescent protein (S65T mutant version); G6PDH, glucose-6-phosphate dehydrogenase; NTG, 1-methyl-3-nitro-1-nitrosoguanidine; PEX, peroxin; PI3-kinase, phosphoinositide 3-kinase; PTS, peroxisomal targeting signal; SD, synthetic/dextrose; SM, synthetic/methanol; SE, synthetic/ethanol; SKL, serine-lysine-leucine; YPD, yeast extract/peptone/dextrose; YND, yeast nitrogen-base/dextrose; YNM, yeast nitrogen-base/methanol.
Y. Sakai was supported by Uehara Memorial Foundation and scholarship from the Ministry of Education, Science, Sports and Culture, Japan. A. Koller was supported by a post-doctoral fellowship from the Swiss National Foundation (no. 8230-046677). This work was supported by an National Institutes of Health grant DK41737 to S. Subramani.
Address all correspondence to Suresh Subramani, Department of Biology, Room 3230 Bonner Hall, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0322. Tel.: (619) 534-2327. FAX: (619) 534-0053. E-mail: ssubramani{at}ucsd.edu
Y. Sakai and A. Koller contributed equally to the manuscript.
Y. Sakai was on sabbatical leave from the Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan.

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-
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-
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-
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[Full Text]
-
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-
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-
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-
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[Full Text]
-
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[Abstract]
[Full Text]
-
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[Full Text]
-
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-
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[Full Text]
-
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(1999). Pex19p Interacts with Pex3p and Pex10p and Is Essential for Peroxisome Biogenesis in Pichia pastoris. Mol. Biol. Cell
10: 1745-1761
[Abstract]
[Full Text]
-
Kim, J., Dalton, V. M., Eggerton, K. P., Scott, S. V., Klionsky, D. J.
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10: 1337-1351
[Abstract]
[Full Text]
-
Hutchins, M., Veenhuis, M, Klionsky, D.
(1999). Peroxisome degradation in Saccharomyces cerevisiae is dependent on machinery of macroautophagy and the Cvt pathway. J. Cell Sci.
112: 4079-4087
[Abstract]
-
Bellu, A. R., Komori, M., van der Klei, I. J., Kiel, J. A. K. W., Veenhuis, M.
(2001). Peroxisome Biogenesis and Selective Degradation Converge at Pex14p. J. Biol. Chem.
276: 44570-44574
[Abstract]
[Full Text]
-
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