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Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells
Published on: February 18, 2014
Structure of proteins in eukaryotic compartments.
Karl Bertrand1, Sergey Reverdatto, David S Burz
1Department of Chemistry, State University of New York at Albany, Albany, New York 12222, United States.
Journal of the American Chemical Society
|July 5, 2012
Summary
Studying protein structure in yeast Pichia pastoris using in-cell NMR revealed that metabolic changes affect protein dynamics. Protein sequestration into storage vesicles broadens NMR spectra, impacting atomic-resolution studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- In-cell Nuclear Magnetic Resonance (NMR) spectroscopy allows atomic-resolution studies of protein structure and dynamics within living cells.
- Metabolic conditions can significantly influence protein behavior and localization within cellular compartments.
Purpose of the Study:
- To investigate the impact of metabolic state on protein structure and dynamics in the yeast Pichia pastoris using in-cell NMR.
- To understand how protein localization within cellular compartments affects NMR spectral quality.
Main Methods:
- Utilized in-cell NMR spectroscopy to analyze protein structure and dynamics in Pichia pastoris.
- Manipulated metabolic conditions by varying carbon sources (dextrose, methanol) and inducing protein overexpression.
- Observed protein localization using microscopy and assessed spectral quality.
Main Results:
- Ubiquitin overexpression in Pichia pastoris under methanol induction resulted in cytosolic localization and well-resolved in-cell NMR spectra.
- Growth on a mixed dextrose-methanol carbon source led to ubiquitin sequestration in small storage vesicles.
- Sequestration into vesicles resulted in broadened in-cell NMR spectra, indicating reduced protein dynamics accessible to small molecules.
- Protein sequestration into vesicles was observed for multiple proteins and yeast strains, suggesting a general cellular mechanism.
Conclusions:
- Metabolic changes in Pichia pastoris influence protein localization and dynamics, detectable by in-cell NMR.
- Sequestration of overexpressed proteins into storage vesicles hinders atomic-resolution in-cell NMR analysis.
- Understanding protein sequestration is crucial for interpreting in-cell NMR data and studying protein behavior in vivo.
Related Concept Videos
01:37Eukaryotic Compartmentalization
One of the distinguishing features of eukaryotic cells is that they contain membrane-bound organelles, such as the nucleus and mitochondria, that carry out specialized functions. Since biological membranes are only selectively permeable to solutes, they help create a compartment with controlled conditions inside an organelle. These microenvironments are tailored to the organelle's specific functions and help isolate them from the surrounding cytosol.
For example, lysosomes in the animal cells...
For example, lysosomes in the animal cells...
01:46Eukaryotic Compartmentalization
One of the distinguishing features of eukaryotic cells is that they contain membrane-bound organelles, such as the nucleus and mitochondria, that carry out specialized functions. Since biological membranes are only selectively permeable to solutes, they help create a compartment with controlled conditions inside an organelle. These microenvironments are tailored to the organelle's specific functions and help isolate them from the surrounding cytosol.
For example, lysosomes in the animal cells...
For example, lysosomes in the animal cells...
01:46Eukaryotic Compartmentalizations
One of the distinguishing features of eukaryotic cells is that they contain membrane-bound organelles, such as the nucleus and mitochondria, that carry out specialized functions. Since biological membranes are only selectively permeable to solutes, they help create a compartment with controlled conditions inside an organelle. These microenvironments are tailored to the organelle's specific functions and help isolate them from the surrounding cytosol.
For example, lysosomes in the animal cells...
For example, lysosomes in the animal cells...
01:45Overview 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...
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...
01:27Globular Proteins
In organisms, proteins are the most abundant macromolecules. They act as the building blocks of life and play various crucial roles in the body. Proteins can be broadly classified into two distinct subtypes based on their shape and solubilities: globular proteins and fibrous proteins.
Globular proteins serve many important physiological functions, such as acting as enzymes, cellular messengers, and molecular transporters. These roles often require the proteins to be soluble in the aqueous...
Globular proteins serve many important physiological functions, such as acting as enzymes, cellular messengers, and molecular transporters. These roles often require the proteins to be soluble in the aqueous...
01:09Golgi Apparatus
Properly folded and assembled proteins are selectively packaged into vesicles that exit the ER. Motor proteins transport these vesicles to the Golgi apparatus for adding modifications that make these proteins functional at their destination.
The Golgi apparatus is a eukaryotic organelle that has a distinctive ribbon-like appearance. It is a primary sorting and dispatch station for cargo arriving from the ER. Newly arriving vesicles enter the cis face of the Golgi, closest to the ER, and are...
The Golgi apparatus is a eukaryotic organelle that has a distinctive ribbon-like appearance. It is a primary sorting and dispatch station for cargo arriving from the ER. Newly arriving vesicles enter the cis face of the Golgi, closest to the ER, and are...