Contact-ID, a tool for profiling organelle contact sites, reveals regulatory proteins of mitochondrial-associated membrane formation

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 117; no. 22; pp. 12109 - 12120
• Main Authors: Kwak, Chulhwan, Shin, Sanghee, Park, Jong-Seok, Jung, Minkyo, Nhung, Truong Thi My, Kang, yeong-Gyun, Lee, Chaiheon, Kwon, Tae-Hyuk, Park, Sang Ki, Mun, Ji Young, Kim, Jong-Seo, Rhee, Hyun-Woo
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 02-06-2020
• Subjects: Biological Sciences; Biotinylation; Calcium; Calcium - metabolism; Calcium transport; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Humans; Membrane proteins; Membrane Proteins - metabolism; Membranes; Mitochondria; Mitochondria - metabolism; Mitochondrial Membranes - metabolism; Organelle Biogenesis; Physical Sciences; Proteins; Proteome - analysis; Proteome - metabolism; Proteomes; Regulatory proteins; Signal Transduction; Tacrolimus Binding Proteins - metabolism; Topology; membrane contact site; FKBP8; proximity labeling; mitochondria-associated membrane (MAM); membrane protein topology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1916584117

The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle–membrane contact sites in live cells.