Hepatic membrane proteins involved in ribosome binding: identification by three procedures

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Ribosome binding to the endoplasmic reticulum (ER) is essential for the synthesis of proteins destined for secretion or insertion into membranes.
• The precise molecular mechanisms and specific proteins involved in anchoring ribosomes to the ER membrane are not fully understood.

Purpose of the Study:

• To identify and characterize non-ribosomal proteins associated with rat liver ribosomes.
• To investigate the role of these associated proteins in the reattachment of ribosomes to the ER membrane in vitro.
• To determine if these proteins are part of the ribosome receptor sites on the ER.

Main Methods:

• Isolation of rat liver ribosomes from rough ER using non-ionic detergent and KCl.
• Extraction of associated non-ribosomal proteins using deoxycholate or detergents at higher KCl concentrations.
• Analysis of extracted proteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE).
• In vitro reattachment assays of ribosomes to degranulated ER membranes.
• Labeling of degranulated membranes with radioactive succinic anhydride.

Main Results:

• Several discrete polypeptides (approx. MW 166,000, 107,000, 100,000, 65,000, and 36,000) were identified in ribosome-associated protein extracts.
• Ribosomes associated with these membrane-derived proteins showed reduced reattachment to degranulated ER membranes in vitro.
• Extraction of similar proteins from degranulated ER with 1 M urea also impaired ribosome reattachment.
• Labeling studies suggested the involvement of specific membrane proteins in ribosome attachment sites.

Conclusions:

• Certain membrane proteins are likely integral components of the ribosome receptor sites on the rat liver endoplasmic reticulum.
• These identified proteins play a significant role in mediating the binding of ribosomes to the ER membrane.