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Originally published as MBC in Press, 10.1091/mbc.E07-09-0868 on September 3, 2008

Vol. 19, Issue 11, 4888-4899, November 2008

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The Membrane Dynamics of Pexophagy Are Influenced by Sar1p in Pichia pastoris

Laura A. Schroder, Michael V. Ortiz, and William A. Dunn, Jr.

Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610-0235

Submitted September 6, 2007; Revised August 8, 2008; Accepted August 21, 2008
Monitoring Editor: Suresh Subramani

Several Sec proteins including a guanosine diphosphate/guanosine triphosphate exchange factor for Sar1p have been implicated in autophagy. In this study, we investigated the role of Sar1p in pexophagy by expressing dominant-negative mutant forms of Sar1p in Pichia pastoris. When expressing sar1pT34N or sar1pH79G, starvation-induced autophagy, glucose-induced micropexophagy, and ethanol-induced macropexophagy are dramatically suppressed. These Sar1p mutants did not affect the initiation or expansion of the sequestering membranes nor the trafficking of Atg11p and Atg9p to these membranes during micropexophagy. However, the lipidation of Atg8p and assembly of the micropexophagic membrane apparatus, which are essential to complete the incorporation of the peroxisomes into the degradative vacuole, were inhibited when either Sar1p mutant protein was expressed. During macropexophagy, the expression of sar1pT34N inhibited the formation of the pexophagosome, whereas sar1pH79G suppressed the delivery of the peroxisome from the pexophagosome to the vacuole. The pexophagosome contained Atg8p in wild-type cells, but in cells expressing sar1pH79G these organelles contain both Atg8p and endoplasmic reticulum components as visualized by DsRFP-HDEL. Our results demonstrate key roles for Sar1p in both micro- and macropexophagy.

This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09-0868) on September 3, 2008.

Address correspondence to: William A. Dunn, Jr. (dunn{at}ufl.edu)

Abbreviations used: AOX, alcohol oxidase; COPII, coat protein complex II; CPY, carboxypeptidase Y; p1CPY, endoplasmic reticulum-associated precursor of CPY; p2CPY, Golgi-associated precursor of CPY; mCPY, vacuole-associated mature carboxypeptidase Y; ER, endoplasmic reticulum; FM4-64, N-(triethlyammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide; MIPA, micropexophagy-specific membrane apparatus; PAS, preautophagosome structure; PI4P, phosphatidylinositol 4'-monophosphate; PE, phosphatidylethanolamine; PVS, perivacuolar structure; SM, sequestering membrane; TCA, trichloroacetic acid; and v-SNARE, vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor.






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