Defunctionalizing intracellular organelles such as mitochondria and peroxisomes with engineered phospholipase A/acyltransferases

Abstract

Organelles vitally achieve multifaceted functions to maintain cellular homeostasis. Genetic and pharmacological approaches to manipulate individual organelles are powerful in probing their physiological roles. However, many of them are either slow in action, limited to certain organelles, or rely on toxic agents. Here, we design a generalizable molecular tool utilizing phospholipase A/acyltransferases (PLAATs) for rapid defunctionalization of organelles via remodeling of the membrane phospholipids. In particular, we identify catalytically active PLAAT truncates with minimal unfavorable characteristics. Chemically-induced translocation of the optimized PLAAT to the mitochondria surface results in their rapid deformation in a phospholipase activity dependent manner, followed by loss of luminal proteins as well as dissipated membrane potential, thus invalidating the functionality. To demonstrate wide applicability, we then adapt the molecular tool in peroxisomes, and observe leakage of matrix-resident functional proteins. The technique is compatible with optogenetic control, viral delivery and operation in primary neuronal cultures. Due to such versatility, the PLAAT strategy should prove useful in studying organelle biology of diverse contexts.

Autophagy Requirements for Eye Lens Differentiation and Transparency

“…Alterations in the membrane structure documented in electron micrographs included rupture of the outer mitochondrial membrane, leaving exposed edges, and internal swelling and disruption of cristae [58]. These observations were supported by a study using HeLa cells in a culture treated with PLAAT3, which showed similar damage to the mitochondria, endoplasmic reticulum, and other organelles [59]. Such distinctive changes to the membranes were not observed in chick embryo or adult Galago (bush baby) lenses where high-resolution electron micrographs were available from the epithelium through the OFZ [86,87].…”

“…Another related finding is the link between phospholipase A/acyltransferases (PLAATs) [57] and the elimination of lens organelles [58]. The translocation of PLATT enzymes to the phospholipid membranes of mitochondria causes the deformation of the mitochondrial surface and the subsequent loss of mitochondrial membrane potential [59]. While depolarization of the mitochondria is not induced when PI3K activity is suppressed [43], the mitochondria become depolarized during lens development just prior to the time that they are delivered to the autophagosomes for elimination [60].…”

Membranes that make fat: roles of membrane lipids as acyl donors for triglyceride synthesis and organelle function

“…PLAAT3 was reported to degrade organelle membranes at the terminal stages of lens development [58]. Moreover, engineered versions of PLAAT members can disrupt mitochondria and peroxisomes in vivo [59]. Interestingly, PLAATs show significant sequence homology to LRATs that, as mentioned earlier, display phospholipid‐dependent acyltransferase activity [60].…”