Mieap forms membrane-less organelles to compartmentalize and facilitate cardiolipin metabolism

Abstract

Liquid droplets function as membraneless organelles that compartmentalize and facilitate efficient biological reactions. They are formed by proteins with an intrinsically disordered region(s) (IDR) via liquid-liquid phase separation. Mieap/SPATA18, a p53-inducible protein, plays a critical role in the suppression of human and murine colorectal tumors via mitochondrial quality control. However, the regulatory mechanism underlying this process remains unclear. Here, we report that Mieap is an IDR-containing protein that drives the formation of liquid droplets in the mitochondria. Mieap liquid droplets (MLDs) specifically phase separate the mitochondrial phospholipid cardiolipin. Lipidomic analysis of cardiolipin suggested that Mieap promotes enzymatic reactions involved in cardiolipin metabolism, including biosynthesis and remodeling. Accordingly, four cardiolipin biosynthesis enzymes, TAMM41, PGS1, PTPMT1, and CRLS1, and two remodeling enzymes, PLA2G6 and TAZ, are phase separated by MLDs. Mieap-deficient mice exhibited altered cristae structure in the liver and kidney mitochondria and a trend of obesity. These results suggest that Mieap drives the formation of membraneless organelles to compartmentalize and promotes cardiolipin metabolism at the inner mitochondrial membrane, thus playing a possible role in mitochondrial quality control.

Identification of a mitochondrial targeting sequence in cathepsin D and its localization in mitochondria

“…For construction of plasmids containing C-terminal enhanced green fluorescent protein-CTSD (pEGFP-CTSD), the nucleotide sequence of CTSD was PCR-amplified using the primers, CTSD-F1 and CTSD-R1. PCR products were digested with Nhe I and Hind III and ligated into pC-EGFP [19] cut with the same enzymes.…”

“…and CTSD-R1. PCR products were digested with Nhe I and Hind III and ligated into pC-EGFP [19] cut with the same enzymes.…”

“…To date, the process of lysosomal localization of CTSD has been characterized as follows [3,4]. Human CTSD, comprising 412 amino acids (pre-pro-CTSD), is translocated into endoplasmic reticulum (ER) via the ER signal peptide (amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], which is removed during translocation to yield pro-CTSD (392 amino acids, 52 kDa) [3]. After undergoing N-linked glycosylation (amino acids 134 and 263) in ER and subsequent modifications in Golgi [5,6], pro-CTSD is transported to the endolysosomal compartment via mannose-6-phosphate (M6P)-dependent or independent pathways [4,7].…”