Related Experiment Video

Updated: Jun 2, 2026

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes
05:57

Peroxisome Staining in Mammalian Cells Using Peroxisome-Specific Probes

Published on: December 19, 2025

Cell-free sorting of peroxisomal membrane proteins from the endoplasmic reticulum

Gaurav Agrawal1, Saurabh Joshi, Suresh Subramani

  • 1Section of Molecular Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0322, USA.

Proceedings of the National Academy of Sciences of the United States of America
|May 18, 2011
PubMed

Insights

Researchers developed a cell-free assay to study how peroxisomal membrane proteins (PMPs) bud from the endoplasmic reticulum (ER). This method identified key requirements for PMP transport, offering a new platform for understanding peroxisome biogenesis.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Peroxisomal membrane proteins (PMPs) are transported to peroxisomes via the endoplasmic reticulum (ER).
  • PMPs assemble in a specialized ER domain, the preperoxisomal ER, before budding.
  • The exact mechanism and biochemical requirements for PMP budding from the ER remain largely unknown.

Purpose of the Study:

  • To establish a cell-free system to investigate the in vitro budding of PMPs from the ER.
  • To identify the biochemical factors and conditions necessary for PMP vesicle formation.
  • To analyze the composition and properties of ER-budded vesicles containing PMPs.

Main Methods:

  • Developed an in vitro cell-free ER-budding assay using the yeast Pichia pastoris.
  • Tracked the ER exit and copackaging of endogenous PMPs, Pex11p and Pex3p, in budded vesicles.
  • Utilized Nycodenz gradients to isolate and analyze ER-budded vesicles.

Main Results:

  • PMP budding from the ER was dependent on ATP, temperature, cytosol, and the protein Pex19p.
  • ER-budded vesicles contained an incomplete set of PMPs, indicating selective packaging.
  • Pex11p budding occurred independently of Pex3p, suggesting distinct transport pathways or intermediates.

Conclusions:

  • The study provides the first biochemical platform to study PMP budding from the ER in vitro.
  • Identified essential factors for PMP vesicle formation, including ATP, cytosol, and Pex19p.
  • Revealed potential distinct mechanisms for the transport of different PMPs, like Pex11p and Pex3p.

More Related Videos

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells08:24

Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells

Published on: August 18, 2017

Related Concept Videos

Protein Import into the Peroxisomes01:27

Protein Import into the Peroxisomes

Cells contain membrane-bound organelles called peroxisomes that oxidize organic molecules by transferring hydrogen atoms to oxygen, producing hydrogen peroxide. Peroxisomes enzymatically convert the released hydrogen peroxide into water and oxygen.
Peroxisomal Protein Import:
Peroxisomes lack the genetic machinery required to code for their own proteins. Hence, most peroxisomal membrane, lumenal and transmembrane proteins are synthesized in the cytoplasm or ER and transported to the peroxisome...
Peroxisomes01:24

Peroxisomes

Peroxisomes are specialized organelles present in fungi, plant, and animal cells. It can vary in number, size, morphology, and activity depending on the type of tissue and the nutritional state of the cell. For example, cells with active lipid metabolism, such as adipocytes, neurons, and hepatocytes, have more peroxisomes than other cells in the body. Besides their primary role in breaking down complex organic molecules, peroxisomes can also synthesize specific macromolecules and participate in...
Peroxisomes01:30

Peroxisomes

Peroxisomes and mitochondria are two important oxygen-utilizing organelles in eukaryotic cells. Mitochondria carry out cellular respiration—the process that converts energy from food into ATP. Peroxisomes carry out a variety of functions, primarily breaking down different substances, such as fatty acids.The peroxisome is a single membrane-bound cellular organelle that can perform several different functions, including lipid metabolism and chemical detoxification. The enzymes within peroxisomes...
Mitochondrial Protein Sorting01:39

Mitochondrial Protein Sorting

Mitochondria are double-membrane organelles of the eukaryotes involved in cellular metabolism, signaling, ATP synthesis, and programmed cell death.  Each of these processes requires specific proteins and enzymes that must be correctly sorted to the right mitochondrial subcompartment for the proper functioning of the organelle.
Most of these mitochondrial proteins are encoded by the nucleus and imported to the mitochondria as unfolded or loosely folded precursors. Mitochondrial precursors...
Overview of Protein Sorting and Transport01:45

Overview of Protein Sorting and Transport

Eukaryotic cells have different membrane-bound organelles with distinct protein requirements. The process by which proteins are targeted to a specific organelle is called protein sorting.
Protein sorting can be of two types: signal-based sorting and vesicle-based trafficking. In signal-based sorting, specific amino acid sequences called sorting signals target proteins to the proper location inside the cell either via gated transport or by protein translocation.  In gated transport, folded...
The Early Endosome: Endocytosis of Transferrin01:28

The Early Endosome: Endocytosis of Transferrin

Essential proteins such as insulin or low-density lipoprotein (LDL) and micronutrients such as iron enter a eukaryotic cell through receptor-mediated endocytosis. Subsequently, the early endosomes fuse with the vesicles containing such receptor-ligand complexes and play a vital role in sorting the incoming ligands and receptors. While the ligands are either degraded inside the vesicle or released into the cytosol, their receptors are returned to the plasma membrane for further rounds of...