Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP

Abstract

<jats:p>A single nuclear gene can be translated into a dual localized protein that distributes between the cytosol and mitochondria. Accumulating evidences show that mitoproteomes contain lots of these dual localized proteins termed echoforms. Unraveling the existence of mitochondrial echoforms using current GFP (Green Fluorescent Protein) fusion microscopy approaches is extremely difficult because the GFP signal of the cytosolic echoform will almost inevitably mask that of the mitochondrial echoform. We therefore engineered a yeast strain expressing a new type of Split-GFP that we termed Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP). Because one moiety of the GFP is translated from the mitochondrial machinery while the other is fused to the nuclear-encoded protein of interest translated in the cytosol, the self-reassembly of this Bi-Genomic-encoded Split-GFP is confined to mitochondria. We could authenticate the mitochondrial importability of any protein or echoform from yeast, but also from other organisms such as the human Argonaute 2 mitochondrial echoform.</jats:p>

The ER protein Ema19 facilitates the degradation of nonimported mitochondrial precursor proteins

“…A number of recent studies reported alternative approaches to follow the import reaction which resulted in surprising observations: (1) Ribosome profiling revealed that cytosolic chaperones and the signal recognition particle play crucial roles in distinguishing mitochondrial and secretory proteins already at very early steps in their synthesis (Schibich et al, 2016;Doring et al, 2017;Costa et al, 2018); (2) proximity labeling suggested that some mitochondrial proteins, in particular hydrophobic inner membrane proteins, explore the mitochondrial surface already during their synthesis (Jan et al, 2014;Williams et al, 2014; Vardi-Oknin and Arava, 2019; Wang et al, 2019) and that, in vivo, many (if not most) mitochondrial surface proteins are in direct proximity to the ER (Hung et al, 2017;Cho et al, 2020); (3) systematic screens of GFP-tagged protein libraries showed that many mitochondrial proteins are prone to accumulate in non-mitochondrial locations under certain growth conditions, in particular on the ER and within the nucleus (Vitali et al, 2018;Backes et al, 2020;Saladi et al, 2020;Shakya et al, 2020;Xiao et al, 2020) and, maybe even more surprising, observed non-mitochondrial residents in mitochondria (Ruan et al, 2017;Bader et al, 2020); and (4) genetic screens reported a very close cooperation of the mitochondrial and ER surface in protein biogenesis (Kornmann et al, 2009;Papic et al, 2013;Okreglak and Walter, 2014;Gamerdinger et al, 2015;Wohlever et al, 2017;Hansen et al, 2018;Vitali et al, 2018;Dederer et al, 2019;Matsumoto et al, 2019). Thus, in vivo, the surfaces of the ER and of mitochondria apparently vividly cooperate to sort proteins to the correct intracellular location.…”

“…, 2020 ) and, maybe even more surprising, observed nonmitochondrial residents in mitochondria ( Ruan et al. , 2017 ; Bader et al. , 2020 ); and 4) genetic screens reported a very close cooperation of the mitochondrial and ER surface in protein biogenesis ( Kornmann et al.…”

Localization and RNA Binding of Mitochondrial Aminoacyl tRNA Synthetases

“…This suggest that weak MTS can complement functions in both compartments [ 56 ]. The recently developed BiG-Mito split GFP approach is likely to allow detection of such weak and unexpected MTS sequences, hence expand the list of aaRSs that are dually localized [ 59 ].…”

“…Notably, overexpression of the cytosolic variant improved growth under respiratory conditions [58]. Moreover, a Bi-genomic split GFP (BiG-split GFP) system was recently developed, in which the cytosolic aaRS was fused to a GFP fragment, while the rest of GFP was expressed inside the mitochondria; a clear mitochondrial signal was detected when cytosolic HisRS was tested [59]. This further emphasizes a possible role in mitochondria targeting for a weak MTS that is present in the cytosolic variant.…”

“…While mitochondrial tRNA Glu is charged by a mitochondria-exclusive GluRS (encoded by the yeast MSE1 gene), it was found that small amounts of the cytosolic GluRS (encoded by GUS1) can enter the mitochondria by weak targeting sequences pinpointed to the first 30 amino acids of the protein [59] or amino acids 190-199 [81]. The imported cytosolic GluRS can mischarge tRNA Gln with Glu, and this serves as a template for a transamidation reaction that yields a Gln-tRNA Gln molecule (of which no dedicated mitochondrial synthetase is known [82,83].…”