Researchers believe that the defective transport of proteins can be linked with diseases such as Alzheimer’s. Prof. Dr. Elke Deuerling and Dr. Martin Gamerdinger, molecular biologists from the University of Konstanz, have now discovered what is necessary to prevent erroneous protein transport.
Proteins are the building blocks of life. The production and transport of proteins in the cells follows complex patterns. Specific signals in the proteins and transport factors ensure that proteins arrive safely at their intended destinations, for example the endoplasmic reticulum (ER) or the mitochondria. A team of researchers headed up by Professor Elke Deuerling in the Department of Molecular Microbiology at the University of Konstanz, has now discovered that successful protein transport and correct sorting requires not only the signal recognition particle (SRP), which stimulates the transport of specific proteins to the ER, but also the nascent polypeptide-associated complex (NAC). Little has previously been known about the role of NAC in the cell. "Many different functions have been postulated for NAC, including a role in the co-translational transport of proteins into the ER. However, data have been quite contradictory," says Prof. Deuerling, speaking of previous scientific knowledge of NAC. Together with Dr. Martin Gamerdinger, Prof. Deuerling has elucidated the effect and importance of the complex in protein sorting and transport.
Elke Deuerling and her team of researchers are concentrating on studying ribosome-associated chaperones, to which NAC also belongs. Their main interest is in elucidating the function of this complex at the ribosomes. Ribosomes generally have a high intrinsic affinity to the translocation pore of the ER membrane, regardless of whether they are active or whether they synthesise a protein with a signalling sequence required for the specific SRP-mediated transport of proteins into the ER. The researchers assumed that this could only be achieved by an inhibitory process that prevented unspecific ribosome binding. "We wanted to find out whether NAC had this particular function and how it affects cellular transport processes," said Prof. Deuerling.
Dr. Gamerdinger decided to study the function of the NAC protein complex in the animal model. It soon became evident that NAC played a crucial role in regulating protein transport. "Simultaneously lowering the expression of the NAC and SRP genes provided the answer that the scientists were seeking. "It led to a greatly potentiated ER stress response, and it showed us that NAC has a decisive effect on the transport of proteins to the ER," said Dr. Martin Gamerdinger.
Dr. Gamerdinger used an experimental set-up with Caenorhabditis elegans (C. elegans) as an experimental animal to show that NAC exerts an important regulatory function in the co-translational transport of proteins to the ER. "We chose C. elegans as a model organism because it cannot survive without NAC. Therefore, we simply used RNA interference methods to reduce the quantity of NAC in adult animals," said Prof. Deuerling. This experimental approach helped the researchers to study NAC-related defects in living animals on the molecular level as well as using biochemical methods. "Another reason for choosing C. elegans was that adult C. elegans is a post-mitotic organism that does not undergo programmed cell death in stress situations. We were thus able to precisely analyse the primary effects of NAC depletion," said Dr. Gamerdinger.
The absence of NAC leads to stress in the ER. This happens because ribosomes tend to bind unspecifically to the ER membrane. Without NAC, the proteins produced by the ribosomes therefore mistakenly end up in the ER as well. The researchers have shown that this is particularly true for proteins intended for the mitochondria. The misdirection of mitochondrial proteins in particular, attracted the researchers' attention as previous studies had already suggested that NAC was somehow involved in protein transport. "Our study has now shown that NAC prevents the erroneous transport of non-authentic proteins into the ER, thus indirectly promoting the transport of these proteins into the mitochondria," says Dr. Gamerdinger highlighting his group's findings. They have shown that animals with low NAC levels only live half as long as animals with normal NAC levels. This therefore revealed that ER and mitochondrial stress are the result of erroneous ribosome transport to the ER, as NAC is no longer there to inhibit the transport of non-authentic proteins into the ER.
Different techniques had to be applied in order to demonstrate the function of NAC. "The application of different methods was the only way that we could obtain a comprehensive picture and make clear statements about the processes," said Prof. Deuerling. The researchers used high-resolution confocal fluorescence microscopy to identify proteins that ended up at the wrong place, i.e. the ER lumen rather than in the mitochondria. Bioformatic approaches were used to analyse system-wide data. "Using these two approaches helped us to find out that NAC mainly prevented cytosolic ribosomes (which translate proteins without an ER signalling sequence) from adhering to the ER membrane, while ribosomes that synthesise ER-specific proteins arrived at their intended ER destination largely unaffected by NAC, and helped only by the SRP," said Dr. Gamerdinger.
The study focuses on aspects of basic research and aims to find out why the NAC is essential and which cellular processes are affected by the complex. However, the study has also come up with many interesting application-related aspects. "We have seen that animals with low NAC levels only live half as long as those with normal NAC levels. Other researchers have also observed that Alzheimer's patients have lower than normal NAC levels. However, whether NAC has a direct effect on the pathogenesis of Alzheimer's is not yet known and needs to be explored further," said Prof. Deuerling.
The next step is therefore to carry out a structural analysis in order to study how the interplay of NAC and SRP affects the sorting of proteins on the mechanical level. "We want to find out how NAC sits on the ribosome and prevents proteins from binding to the ER translocation pore," says Dr. Gamerdinger, referring to future research. "We will also explore whether the function of NAC decreases with age and under protein stress, which, amongst other things leads to Alzheimer's and other diseases," concluded Prof. Deuerling.