Related Experiment Video
Updated: Mar 30, 2026
07:07
Evaluation of Protein–Protein Interactions using an On-Membrane Digestion Technique
Published on: July 19, 2019
7.2K
Extracellular protease digestion to evaluate membrane protein cell surface localization.
Richard N Besingi1, Patricia L Clark1,2
1Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana, USA.
Nature Protocols
|November 20, 2015
Summary
This study presents a method to assess membrane protein secretion in Gram-negative bacteria. It highlights the importance of selecting the right reporter protein to accurately measure membrane protein localization and ensure membrane integrity.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Membrane proteins are vital for cellular signaling and display on the cell surface.
- Assessing membrane protein secretion requires verifying membrane integrity during protease digestion experiments.
- Current methods may lack accuracy due to improper selection of control elements.
Purpose of the Study:
- To describe a protocol for evaluating the efficiency of protein secretion to the outer surface of Gram-negative bacteria.
- To emphasize the critical role of intracellular reporter proteins in confirming membrane integrity.
- To provide a reliable method for accurate membrane protein localization studies.
Main Methods:
- Utilizing extracellular protease digestion to assess membrane protein secretion.
- Employing intracellular reporter proteins (e.g., SurA) sensitive to cell lysis to confirm membrane integrity.
- Standard biochemistry techniques including cell lysis, gel electrophoresis, and western blotting.
Main Results:
- The choice of reporter protein significantly impacts the accuracy of membrane protein localization measurements.
- Protease-resistant reporters (e.g., maltose-binding protein) yield inaccurate results.
- Protease-sensitive reporters (e.g., SurA) provide sensitive and accurate indicators of membrane integrity.
Conclusions:
- This protocol offers a robust method for determining membrane protein secretion efficiency in Gram-negative bacteria.
- Careful selection of reporter proteins is essential for validating membrane integrity and achieving accurate localization data.
- The approach is adaptable for evaluating membrane protein localization in eukaryotic cells and organelle membranes.
Related Concept Videos
01:19Translocation of Proteins into the Mitochondria
13.7K
Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...
13.7K
02:18The Proteasome
10.5K
Eukaryotic cells can degrade proteins through several pathways. One of the most important amongst these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. A series of enzymes carry out the ubiquitination of the target proteins - E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
10.5K
01:13The Proteasome
2.0K
Eukaryotic cells can degrade proteins through several pathways. One of the most important among these is the ubiquitin-proteasome pathway. It helps the cell eliminate the misfolded, damaged, or unwarranted cytoplasmic proteins in a highly specific manner.
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...
2.0K
01:23Role of Matrix Metalloproteases in Degradation of ECM
3.7K
Matrix metalloproteases (MMPs) are enzymes involved in the hydrolysis of proteins and glycoproteins of the extracellular matrix. MMPs are essential for the migration and proliferation of cells through the dense matrix network, throughout embryonic development, and throughout morphogenesis. The first MMP activity discovered was a collagenase in a tadpole's tail undergoing metamorphosis. The active collagen deposition and modifications lead to the morphogenesis of tadpoles into the adult...
3.7K
01:18Detergent Purification of Membrane Proteins
6.8K
Detergents are used to purify the integral proteins of the membrane. The hydrophobic portion of the detergent can replace membrane phospholipids while solubilizing the membrane proteins. When detergent monomers reach a specific concentration in a solution called critical micelle concentration (CMC), they form micelles. Above CMC, the concentration of the detergent monomers remains in equilibrium with the micelle. The number of detergent monomers present in the CMC varies for each detergent, and...
6.8K
01:17The Proteasome Structure
2.2K
The ubiquitin-proteasome pathway is a well-known mechanism utilized by eukaryotic cells to remove cytoplasmic proteins that are misfolded, damaged, or no longer needed. In this pathway, the protein that needs to be eliminated undergoes a process called ubiquitination, where a chain of ubiquitin molecules is attached to the 48th lysine residue of the target protein. This ubiquitin modification helps the proteasome distinguish between a target protein and a healthy protein.
The proteasome is an...
The proteasome is an...
2.2K