Related Experiment Videos
Synthesis of intracellular membrane proteins in vitro. Relation between rough endoplasmic reticulum and mitochondrial
Abstract:
Hepatic rough microsomes were incubated in a messenger-dependent protein-synthesizing system from rabbit reticulocytes. Up to 30% of the total product labelled with [35S]methionine, and subsequently recovered with the microsomes, was located in an intrinsic protein fraction associated with these membranes, i.e. was retained by the membrane following extensive sonication in the presence of 1.5 M KCl, 0.1% deoxycholate, and 5 mM ethylenediaminetetra-acetate (EDTA). When products synthesized with the use of membrane-free mRNA from rough microsomes and free polysome were post-incubated with rough microsomes, ribosome-stripped rough microsomes, or outer mitochondrial membrane, low amounts of intrinsic-type polypeptide product were recovered with these membranes. Higher recovery was achieved, however, when ribosome-stripped rough microsomes were added at the beginning of polypeptide synthesis in a reticulocyte lysate supplemented with additional ribosomal-wash factors. Analysis of these products by polyacrylamide gel electrophoresis showed that a number co-migrated with intrinsic proteins located in both rough microsomes and mitochondrial outer membrane. In addition, a prominent in vitro product co-migrated with a major protein which is located in outer mitochondrial membrane fractions, but is barely detectable in rough microsomal fractions. The present experiments were unable to detect a unique set of intrinsic polypeptides which were synthesized and assembled in vitro under the direction of mRNA from free polysomes, and not from rough microsomes. The results suggest that synthesis of at least some intrinsic membrane proteins which are destined for the outer mitochondrial membrane occurs on rough ER in rat liver.
Insights
Rat liver rough endoplasmic reticulum (RER) synthesizes intrinsic membrane proteins. Some proteins destined for the outer mitochondrial membrane are synthesized on the RER, not free polysomes.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Intrinsic membrane proteins are crucial for cellular function.
- Understanding the synthesis and localization of these proteins is key to cellular processes.
- The role of rough endoplasmic reticulum (RER) in synthesizing proteins destined for other organelles is an area of active research.
Purpose of the Study:
- To investigate the site of synthesis for intrinsic membrane proteins, particularly those of the outer mitochondrial membrane.
- To determine if rough microsomes or free polysomes are responsible for synthesizing these proteins.
- To elucidate the role of the RER in the biogenesis of mitochondrial outer membrane proteins.
Main Methods:
- Incubation of hepatic rough microsomes in a rabbit reticulocyte protein-synthesizing system.
- Labeling of newly synthesized proteins with [35S]methionine.
- Fractionation of membrane-associated proteins using sonication, KCl, deoxycholate, and EDTA.
- Post-incubation and co-incubation experiments with various membrane fractions and mRNA.
- Analysis of protein products using polyacrylamide gel electrophoresis.
Main Results:
- A significant portion (up to 30%) of newly synthesized proteins associated with rough microsomes were identified as intrinsic membrane proteins.
- Post-incubation of synthesized products with membranes yielded low recovery of intrinsic proteins.
- Addition of ribosome-stripped rough microsomes at the start of synthesis increased recovery of intrinsic proteins.
- Synthesized products co-migrated with intrinsic proteins of both rough microsomes and outer mitochondrial membranes.
- A prominent in vitro product matched a major outer mitochondrial membrane protein not readily detected in rough microsomes.
Conclusions:
- The synthesis of at least some intrinsic membrane proteins destined for the outer mitochondrial membrane occurs on the rough endoplasmic reticulum in rat liver.
- This suggests a role for the RER in the biogenesis of mitochondrial outer membrane proteins.
- Free polysomes do not appear to synthesize a unique set of intrinsic polypeptides distinct from those synthesized on rough microsomes.