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Ribosome-membrane interaction. Nondestructive disassembly of rat liver rough microsomes into ribosomal and membranous
Abstract:
In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl(2), 50 mM Tris.HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [(3)H]puromycin into hot acid-insoluble material and from the release of [(3)H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25-100 mM) as at high (500-1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction ( approximately 15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.
Insights
High salt conditions and puromycin treatment can separate rat liver rough microsomes into ribosomes and stripped membranes. This method reveals two distinct ribosome-membrane binding interactions, one direct and one mediated by nascent polypeptide chains.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Rough microsomes are key sites for protein synthesis and translocation.
- Understanding ribosome-membrane interactions is crucial for protein folding and secretion.
Purpose of the Study:
- To develop a method for nondestructively disassembling rough microsomes.
- To elucidate the mechanisms of ribosome-membrane binding.
Main Methods:
- Incubation of rat liver rough microsomes with puromycin in high ionic strength buffer (750 mM KCl).
- Analysis of released ribosomes and stripped membranes for integrity and activity.
- Quantification of nascent polypeptide chain release and association.
Main Results:
- Puromycin treatment at high salt concentrations released up to 85% of bound ribosomes as active subunits.
- Ribosomes bind to membranes via a direct interaction (high salt labile) and nascent chain anchoring (puromycin labile).
- A fraction of ribosomes remained bound and required harsh conditions for release, suggesting diverse binding modes.
Conclusions:
- A puromycin-based method effectively disassembles rough microsomes, separating ribosomes and membranes.
- Ribosome-membrane association involves at least two distinct mechanisms.
- Further investigation is needed to characterize the tightly membrane-bound ribosomes.