Structural Biochemistry/Protein function/Integral Membrane Protein
The basic mechanism of alpha (α) helical membrane proteins being integrated into the endoplasmic reticulum membrane has been well established. However, scientists seek to find clearer details of these mechanisms as well as their kinetics to understand membrane protein integration as a whole. It is therefore important to use in vivo and in vitro experiments to understand more about membrane protein integration.
Mechanism of General Integral Membrane Proteins
The currently proposed mechanism is as follows: Rough endoplasmic reticulum targeting signals, particularly the transmembrane span (TM), are recognized by a signal recognition particle (SRP) in the cytosol. This transmembrane span is attached to the ribosome-nascent polypeptide complex (RNC) which in turn is attached to the SRP; the SRP then forms a stable complex between itself and the endoplasmic reticulum SRP receptor on its membrane. As the SRP dissociates, the RNC attaches to Sec61, a cotranslational translocation channel. Before even passing through this channel, the TM span folds into the α helix orientation. In doing this, there are enough hydrogen bond donors and acceptors for the rest of the protein to adopt the proper α helix folding.
In vitro vs in vivo Experiments
In order to further study the particulars of the aforementioned mechanism, scientists have used in vitro experiments, yet these have problems. One issue is that in vitro experiments do not provide accurate data on the kinetics of membrane protein integration. Obviously, the limiting factor in this case is that in vitro experiments are not in the actual, living cell. In comparison, in vivo studies show that the eukaryotic translation systems synthesize proteins at 5-7 residues per second. In comparison, in vitro experiments are limited to synthesizing only 5-10% of the speed. Another issue is, again, the fact that past experimental data have been received from in vitro environments, unreflective of the actual cells. In this case, the time it took to purify and analyze membrane protein integration intermediates may have resulted in studying the kinetics of what was left after equilibrium of the intermediates, not the actual kinetics of the reactive intermediates.
In vivo experiments have enlightened some details of the mechanism. In one experiment, saccharomyces cerevisiae, or budding yeast, cells were used. While studying the SRP-SR targeting pathway, scientists disrupted the SR and SRP genes. This resulted in a crippled cell growth rate on top of other functional losses, yet the cell adapted to the loss by utilizing an SRP-SP independent pathway. This showed researchers the importance of utilizing in vivo experiments to fully understand how particular mechanisms worked. From this experiment, one could see that the SRP-SR pathway was not the sole way for the RNC to bind to the membrane.
Another in vivo experiment clarified the kinetics of the way RNCs are able to target themselves to the endoplasmic reticulum. In this experiment, the luminal domain of the membrane protein was tagged with a phosphorylation site. Since phosphorylation of the residues could only occur when the kinase was in the cytosol and since the luminal domain was exposed to the cytosol infrequently, scientists were able to calculate the time required for SRP-mediated RNC targeting.
The orientation, or specifically, topology, of TM spans was also studied using in vivo experiments. In normal functioning cells, the topology is determined by the charged residues on the TM span. For this particular cell, there were two topologies: one (type 2) that occurred when there was a net positive charge on the N-terminal and another topology when there was a net positive charge on the C-terminal (type 1). Upon mixing these two topologies in vivo, scientists found that type 2 membrane proteins inserted themselves into the Sec61 complex as the type 1 tomology, but they inverted back to their original type 2 form within 50 seconds. These findings were substantiated by in vitro experiments as well. Scientists also found that this inversion occurred more rapidly when the TM span had positively charged residues in front.
These examples show the importance of in vivo experiments in determining the minute details of the integration of α helical membrane proteins into the endoplasmic reticulum. While it is true that in vitro experiments opened many views into how the mechanism worked, there was a very limited view. In vivo experiments, when combined with in vitro experiments, can provide an unparalleled view of exactly how the mechanism may work as well as how the kinetics of the reactions occur. Furthermore, these two types of experiments, specifically in vivo can open many avenues into other mechanisms and cell functions previously unknown.
- Gilmore, Reid; Mandon, Elisabet C. (2012). "Understanding integration of α-helical membrane proteins: The next steps". Trends in Biochemical Sciences 37 (8): 303–8. doi:10.1016/j.tibs.2012.05.003. PMID 22748693.