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PMID: 8670828 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Characterization of a novel component of the peroxisomal protein import apparatus using fluorescent peroxisomal proteins.

The EMBO journal ·Vol. 15 ·No. 13 ·1996-07-01 ·Pages 3275-85

Kalish JE, Keller GA, Morrell JC, Mihalik SJ, Smith B, Cregg JM, Gould SJ

Abstract

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.

MeSH Terms
Amino Acid Sequence Base Sequence Biological Transport Carrier Proteins/genetics,metabolism Cloning, Molecular Fungal Proteins/genetics,metabolism Green Fluorescent Proteins Luminescent Proteins/metabolism Membrane Proteins/genetics,metabolism Membrane Transport Proteins Microbodies/metabolism Microscopy, Electron Microscopy, Fluorescence Molecular Sequence Data Pichia/metabolism Protein Binding Zinc Fingers/genetics
Chemicals
Carrier Proteins Fungal Proteins Luminescent Proteins Membrane Proteins Membrane Transport Proteins Pas10 protein, Pichia pastoris Green Fluorescent Proteins
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Kalish J E
Department of Biological Chemistry, The John Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Keller G A
Morrell J C
Mihalik S J
Smith B
Cregg J M
Gould S J
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1996-07-01
Pages
3275-85
Language
English
Region
England
NLM ID
8208664
PMCID
PMC451890
Subset
IM
Grants
NIDDK NIH HHS · DK45787 · United States
Databases
GENBANK
U58140
External Links
PubMed source PMC Full Text View journal details
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