We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.
%0 Journal Article
%1 bhartiPEX14RequiredMicrotubulebased2011
%A Bharti, Pratima
%A Schliebs, Wolfgang
%A Schievelbusch, Tanja
%A Neuhaus, Alexander
%A David, Christine
%A Kock, Klaus
%A Herrmann, Christian
%A Meyer, Helmut E.
%A Wiese, Sebastian
%A Warscheid, Bettina
%A Theiss, Carsten
%A Erdmann, Ralf
%C England
%D 2011
%J Journal of cell science
%K & Animals,Cell Binding,Protein Fractions/chemistry/metabolism,Swine,to_read Gel,Humans,Membrane Line Proteins/biosynthesis/genetics/isolation Transport,Repressor Tumor,Chromatography purification/*metabolism,Microtubules/chemistry/genetics/*metabolism,Peroxisomes/chemistry/genetics/*metabolism,Protein purification/*metabolism,Subcellular
%N Pt 10
%P 1759--1768
%R 10.1242/jcs.079368
%T PEX14 Is Required for Microtubule-Based Peroxisome Motility in Human Cells.
%V 124
%X We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.
@article{bhartiPEX14RequiredMicrotubulebased2011,
abstract = {We have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.},
added-at = {2024-05-17T13:01:35.000+0200},
address = {England},
author = {Bharti, Pratima and Schliebs, Wolfgang and Schievelbusch, Tanja and Neuhaus, Alexander and David, Christine and Kock, Klaus and Herrmann, Christian and Meyer, Helmut E. and Wiese, Sebastian and Warscheid, Bettina and Theiss, Carsten and Erdmann, Ralf},
biburl = {https://www.bibsonomy.org/bibtex/208e9713d76641e824a93cbad5066393a/warscheidlab},
doi = {10.1242/jcs.079368},
interhash = {46a3f1ae6ddb10aa7cc28a3a0a39e338},
intrahash = {08e9713d76641e824a93cbad5066393a},
issn = {1477-9137 0021-9533},
journal = {Journal of cell science},
keywords = {& Animals,Cell Binding,Protein Fractions/chemistry/metabolism,Swine,to_read Gel,Humans,Membrane Line Proteins/biosynthesis/genetics/isolation Transport,Repressor Tumor,Chromatography purification/*metabolism,Microtubules/chemistry/genetics/*metabolism,Peroxisomes/chemistry/genetics/*metabolism,Protein purification/*metabolism,Subcellular},
langid = {english},
month = may,
number = {Pt 10},
pages = {1759--1768},
pmid = {21525035},
timestamp = {2024-05-17T13:01:35.000+0200},
title = {{{PEX14}} Is Required for Microtubule-Based Peroxisome Motility in Human Cells.},
volume = 124,
year = 2011
}