Protein Linking Key Organelles May Shed Light on Neurodegenerative Disease Development

Scientists report that they have developed a technique to identify proteins that allow the mitochondria and the endoplasmic reticulum to attach to each other. Faulty connections between the ER and mitochondria have been implicated in several neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases.

The research (“Ascorbate Peroxidase Proximity Labeling Coupled with Biochemical Fractionation Identifies Promoters of Endoplasmic Reticulum Mitochondrial Contacts”) appears in the Journal of Biological Chemistry.

“Here we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs [endoplasmic reticulum (ER)-mitochondria contacts] in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method,” write the investigators.

“We identified 405 proteins with a 2.0-fold cut-off ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified RETICULON1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.”

“Think of [an organelle] like a ferry docking at one site, unloading and loading passengers and cars, and then going to another site and doing the same thing,” said Jeffrey Golden, M.D., a professor at Brigham and Women’s Hospital and Harvard Medical School who oversaw the work. “Their ability to dock, load, and unload cargo requires guides or ramps of specific width and heights that connect the boat and land or they cannot freely load and unload.”

Dr. Golden’s team relied on a technique developed to show contact between proteins. The method takes advantage of APEX, which can attach biotin to nearby proteins. The researchers engineered cells to produce mitochondria that had APEX attached to their outer membranes, and then added biotin to the cells for the APEX to use to label nearby proteins.

The team then isolated parts of the cell that contained the ER, purified those proteins that had biotin attached, and identified the ones found in the ER using mass spectrometry. Because the APEX was attached to mitochondria, only those proteins that came into close proximity to the mitochondria could have had biotin attached. Thus, the biotin served as a signal indicating which proteins had been involved in the ER-mitochondria contact.

“It was previously feasible to only look at one molecule at a time to assess what it interacted with,” Dr. Golden said. “The method we have used is more rapid and allows an unbiased look at a whole system and what’s happening at that organelle’s interface.”

Using this screening approach, the scientists looked at an ER protein called RTN1a, which was previously known to contribute to the ER’s shape. They later confirmed that this protein also helped mitochondria to attach to the ER.

This work raises the possibility that defects in RTN1a could contribute to the problems experienced by patients with neurodegenerative diseases, but the scientists say additional studies, including research in neural cells, are needed.

Dr. Golden speculates that the proteins important for ER–mitochondrial contact might be different in different cell types.

“Does the liver use the same proteins to control these kinds of interactions that neural cells do? Is one [protein] more important for calcium exchange and another set of proteins more important for lipid exchange?” he asked. “I think there’s a lot of cell biology that we just don’t know and could be answered [using this method].”

The team is now using the APEX-mass spectrometry method to compare proteins involved in ER–mitochondrial contacts between normal and patient-derived neural cells.