QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stewart, M. Q. Articles by Goodman, J. M. Search for Related Content PubMed PubMed Citation Articles by Stewart, M. Q. Articles by Goodman, J. M. Social Bookmarking                         What's this?

Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041, USA

View larger version (45K): [in a new window]   Fig. 1. Separation of peroxisomal subcompartments from activated spheroplasts. (A) Outline of procedure. After metabolic labeling, activated spheroplasts are subjected to osmotic lysis at pH 5.5. An organellar pellet (PEL) and supernatant (SUP) are generated by centrifugating the lysate at 20,000 g. (There are no prespins.) The pellet is resuspended in buffer at pH 9.0 to lyse peroxisomes, and then spun at 100,000 g, generating a supernatant containing peroxisomal matrix (MAT) and a pellet containing peroxisomal membrane (MEM). Analysis of markers in a typical experiment is shown in B-D. (B) Formaldehyde dehydrogenase, a cytosolic enzyme. There is little contamination of cytosol in the matrix. (C) Coomassie Blue staining indicates the integrity of peroxisomes after cell lysis, and the subsequent release of matrix. (D) Immunoblot with anti-Pmp47, an integral peroxisomal membrane protein.

View larger version (33K): [in a new window]   Fig. 2. The import of AO and DHAS from cytosol into peroxisomal matrix. Activated spheroplasts were labeled as described and chased for the times indicated. Spheroplasts from each time point were subjected to fractionation. (A) Fractions were immunoprecipitated with antibody to the proteins indicated and subjected to SDS-PAGE and fluorography. (B) Immunoprecipitates were quantified by densitometry. Results were expressed as a ratio of the label in the pellet compared with supernatant and pellet. (C) Sample buffer was added directly to the PEL, MAT and MEM fractions before SDS-PAGE and fluorography.

View larger version (29K): [in a new window]   Fig. 3. Alcohol oxidase is imported as a monomer. The supernatant and matrix fractions from pulse-chased spheroplasts were subjected to velocity sedimentation. Fractions of the sucrose gradients were immunoprecipitated with anti-AO antiserum. BSA and mature AO were used as markers of the monomeric and octameric species. Asterisk represents an AO degradation product. Open arrowhead indicates intermediate AO forms on gradient.

View larger version (17K): [in a new window]   Fig. 4. Dihydroxyacetone synthase is imported as a dimer. Analysis similar to that in Fig. 3 except the gradients were spun for a longer time and fractions were subjected to immunoprecipitation with anti-DHAS antiserum. BSA and mature DHAS served as markers of monomeric and dimeric species.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter