# Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP

Solène Zuttion, Bruno Senger, Chiranjit Panja, Sylvie Friant, Róża Kucharczyk, Hubert Becker

► To cite this version:

Solène Zuttion, Bruno Senger, Chiranjit Panja, Sylvie Friant, Róża Kucharczyk, et al.. Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP. Methods in Enzymology, 2024, Methods in Enzymology, 706, pp.75-95. 10.1016/bs.mie.2024.07.028. hal-05141285

HAL Id: hal-05141285

https://hal.science/hal-05141285v1

Submitted on 3 Jul 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

Public Domain
# Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP

Solène Zutton¹, Bruno Senger¹, Chiranjit Panja², Sylvie Friant¹, Róża Kucharczyk², Hubert Dominique Becker¹

¹Université de Strasbourg, CNRS, Génétique Moléculaire, Génomique, Microbiologie, UMR 7156, 4 Allée Konrad Röntgen, 67084 Strasbourg Cedex, France. ²Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

‡ Correspondence to: h.becker@unistra.fr and roza@ibb.waw.pl

## Abstract

Even if a myriad of approaches has been developed to identify the subcellular localization of a protein, the easiest and fastest way still remains to fuse the protein to Green Fluorescent Protein (GFP) and visualize its location using fluorescence microscopy. However, this strategy is not well suited to visualize the organellar pools of proteins that are simultaneously localized both in the cytosol and in organelles because the GFP signal of cytosolic pool of the protein (cytosolic echoform) will inevitably mask or overlay the GFP signal of the organellar pool of the protein (organellar echoform). To solve this issue, we engineered a dedicated yeast strain expressing a Bi-Genomic Mitochondrial-Split-GFP. This split-GFP is bi-genomic because the first ten β-strands of GFP (GFP<sub>β1-10</sub>) are encoded by the mitochondrial genome and translated by mitoribosomes whereas the remaining β-strand of GFP (GFP<sub>β11</sub>) is fused to the protein of interest encoded by the nucleus and expressed by cytosolic ribosomes. Consequently, If the GFP<sub>β11</sub>-tagged protein localizes into mitochondria, GFP will be reconstituted by self-assembly GFP<sub>β1-10</sub> and GFP<sub>β11</sub> thereby generating a GFP signal restricted to mitochondria and detectable by regular fluorescence microscopy. In addition, because mitochondrial translocases and import mechanisms are evolutionary well conserved, the Bi-Genomic Mitochondrial-Split-GFP yeast strain can be used to probe mitochondrial importability of proteins regardless of their organismal origins and can thus serve to identify unsuspected mitochondrial echoforms readily from any organism.
Key words

Protein import, dual-localized, mitochondria, Split-GFP, *Saccharomyces cerevisiae*,
microscopy

4166 words w/o references and Figure legends

<table><tr><td>1.</td><td>Introduction</td><td>1</td></tr><tr><td>1.1.</td><td>Multi-localized proteins</td><td>1</td></tr><tr><td>1.2.</td><td>Engineering of the BiG Mito-Split-GFP strain by mitochondrial DNA editing</td><td>2</td></tr><tr><td>1.2.1.</td><td>Construction of the RKY112 strain (Mata [ρ<sup>+</sup> atp6::ARG8<sup>m</sup> 5'UTR<sub>COX2</sub> ATP6 3'UTR<sub>COX2</sub>])</td><td>3</td></tr><tr><td>1.2.2.</td><td>Construction of the RKY176 BiG Mito-Split-GFP strain</td><td>4</td></tr><tr><td>1.3.</td><td>β11-chapelet tagging of nuclear-encoded proteins</td><td>5</td></tr><tr><td>1.4.</td><td>Overview of the use of Big Mito-Split-GFP for probing mitochondrial translocation of a protein</td><td>5</td></tr><tr><td>2.</td><td>Materials</td><td>6</td></tr><tr><td>2.1.</td><td>For cloning and engineering of strains</td><td>6</td></tr><tr><td>2.2.</td><td>For cell growth</td><td>7</td></tr><tr><td>2.3.</td><td>For protein immunodetection</td><td>7</td></tr><tr><td>2.4.</td><td>For fluorescence microscopy</td><td>8</td></tr><tr><td>3.</td><td>Methods</td><td>8</td></tr><tr><td>3.1.</td><td>GFP<sub>β11ch</sub> epitope tagging</td><td>9</td></tr><tr><td>3.2.</td><td>Transformation of the BiG Mito-Split-GFP strain</td><td>9</td></tr><tr><td>3.3.</td><td>Growth of the BiG Mito-Split-GFP strain expressing GFP<sub>β11ch</sub>-tagged proteins</td><td>10</td></tr><tr><td>3.4.</td><td>Protein extraction and Western-blot analyses</td><td>10</td></tr><tr><td>3.5.</td><td>Visualizing mitochondrial translocation by fluorescence microscopy</td><td>11</td></tr><tr><td>Acknowledgements</td><td></td><td>12</td></tr><tr><td>References</td><td></td><td>12</td></tr><tr><td>Tables</td><td></td><td>12</td></tr><tr><td>Figure legends</td><td></td><td>12</td></tr></table>
<figure><img src="image_1.png" /></figure>
# 1. Introduction

## 1.1. Multi-localized proteins

Proteins display a remarkable diversity in their functions and subcellular locations. Among them, a fascinating class known as dual-localized proteins has attracted the attention of researchers (Smalheiser, 1996). Dual-localized proteins, as the name suggests, possess the ability to reside in more than one subcellular compartment, challenging traditional notions of protein trafficking and localization. Unlike conventional proteins, which often exhibit strict compartmentalization, dual-localized proteins not only defy such rigid boundaries, straddling multiple locales within the cell but also often perform different roles across the distinct compartments they reach. In particular, proteins with dual localization in the cytoplasm and the mitochondria are of particular importance since they are involved in a myriad of fundamental processes such as organelle communication, signaling cascades, and metabolic regulation (Eisenberg- Bord & Schuldiner, 2017). Unraveling the molecular mechanisms responsible for the simultaneous localization of a single peptide in the cytosol and in mitochondria still represents a formidable challenge. Central to this quest is the identification of cryptic nonconventional targeting sequences or motifs within this class of proteins that govern their multiple subcellular distribution (Yogev & Pines, 2011). These signals may encompass a diverse array of structural motifs, post-translational modifications, or protein-protein interactions that would dictate either their organellar translocation or cytosolic retention (Frechin et al., 2009 ; Frechin et al., 2014). Advances in high-resolution imaging techniques, mass-spectrometry-based proteomic profiling, and computational modeling have revolutionized our ability to inventory full sets of mitochondrial proteins, also called mitoproteomes, across a wide range of organisms (Ben- Menachem et al., 2011 ; Baker et al., 2024). The number of identified dual-localized proteins in these mitoproteome is constantly evolving as new and cutting-edge mass spectrometry approaches are developed (Morgenstern et al., 2017; Vögtle et al., 2017). Yet these cutting-edge methodologies often rest on getting highly pure mitochondrial fractions which is more than exceedingly challenging given the variety of other organellar compartments that make inter-membrane contacts with mitochondria (Murley & Nunnari, 2016). This often casts doubts on the legitimate participation of a protein having other or additional localizations to mitoproteomes.
The most popular method that allows to verify that a given protein can be localized in mitochondria without having to purify mitochondria is to fuse the protein with a fluorescent protein and observe the localization of this chimeric protein using fluorescence microscopy coupled to labeling of mitochondria with specific dyes. This strategy, however, is not well adapted for visualizing dual-localized proteins since the cytosolic pool of a dual-localized protein (called cytosolic echoform) is usually much more abundant than the mitochondrial one (called mitochondrial echoform) and thus this will inevitably mask the fluorescence signal generated by the mitochondrial echoform. To circumvent this technical limitation, we engineered a yeast strain containing a new type of Split-GFP system designated as the Bi-Genomic Mitochondrial-Split-GFP (BiG Mito-Split-GFP) (Bader et al., 2020). This new type of compartment-specific Split-GFP is bi-genomic because the synthetic gene encoding the 10 first β-strands of the GFP ($GFP_{β1-10}$) has been introduced in the mitochondrial genome and thus is translated by mitoribosomes inside the mitochondrial matrix, while the remaining last β-strand of GFP ($GFP_{β11}$) is fused to the nuclear-encoded protein to be tested (either plasmid-borne or chromosomally fused) and thus translated by cytosolic ribosomes. Expressing a $GFP_{β11}$-tagged dual-localized protein (cytosolic and mitochondrial) in the BiG Mito-Split-GFP strain will result in GFP reconstitution only inside mitochondria and thus will only yield GFP signal emission of the $GFP_{β11}$-fused mitochondrial echoform of the dual-localized protein which can be visualized under any type of fluorescence microscope (Figure 1). [Insert Figure 1 here]

## 1.2. Engineering of the BiG Mito-Split-GFP strain by mitochondrial DNA editing

The genomes of *S. cerevisiae*, both nuclear and mitochondrial [mtDNA], undergo homologous recombination (Bonnefoy & Fox, 2002). This feature allows to easily remove sequences of or introduce mutations into a specific gene. The ability of yeast cells to survive without mtDNA is another feature that allows for its modification. Recombination of a DNA fragment into the mitochondrial genome requires that it resides inside the mitochondrial network. This DNA fragment is first cloned into the pUC19-based plasmid. This plasmid must be introduced by the biolistic transformation (Bonnefoy & Fox, 2002) inside the mitochondria of a yeast strain totally lacking mtDNA ($\rho^0$) and bearing *kar1-1* mutation in the nuclear *KAR1* gene, which block the fusion of the nuclei during crossing with another haploid strain (Conde & Fink, 1976). The
mitochondrial transformants need then to be purified from mostly nuclear transformants. Such clones are then crossed with the recipient strain containing mtDNA (ρ⁺). The *kar1-1* mutation blocks the fusion of the nuclei, while the mitochondrial network fuses, two different mtDNAs recombinate and the mitochondrial recombinant may be selected – the process cold cytoduction. Selection of the recombinants is possible thanks to the genetic markers that differ the nuclei of both strains and permit to distinguish between the parental and recombinant mtDNA, explained below.

The aim of the study was to construct respiring strain bearing the *GFP*β₁-₁₀ gene in the fully functional mtDNA. The mtDNA of *S. cerevisiae* encodes only eight peptides which are either components of the respiratory chain complexes (7 of them) or of the mitochondrial ribosomes (Foury et al., 1998). The critical point in development of the mtDNA site-directed mutagenesis, was successful construction of the yeast strain MR10, in which the coding sequence of *ATP6* gene, encoding the subunit 6 of mitochondrial ATP synthase, was replaced with *ARG8*<sup>m</sup> (*atp6::ARG8*<sup>m</sup>) (Rak et al., 2007). The *ARG8*<sup>m</sup> is a mitochondrial version of a nuclear gene (*ARG8*) encoding a mitochondrial protein involved in arginine biosynthesis (Steele et al., 1996). Construction of the BiG Mito-Split-GFP strain was a multiple steps process, described below. It required a yeast respiring strain having the one mitochondrial locus flanked by its endogenous 5'- and 3' regulatory sequences available for integration of any gene sequence. Such a strain – RKY112 – bears in its DNA the *atp6::ARG8*<sup>m</sup> replacement while the *ATP6* gene sequence was integrated in between the regulatory sequences of the COX2 gene at the COX2 locus (Bader et al., 2020).

### 1.2.1. Construction of the RKY112 strain (Mata [ρ⁺ atp6::ARG8<sup>m</sup> 5'UTR<sub>cox2</sub> ATP6 3'UTR<sub>cox2</sub>])

Construction of this strain was a three-step process (Figure 2). [Insert Figure 2 here]. We first constructed a ρ⁺ strain (RKY83) with a complete deletion of *ATP6* coding sequence (*atp6::ARG8*<sup>m</sup>) and a partial deletion in *COX2*, *cox2-62* (Mata [ρ⁺ *atp6::ARG8*<sup>m</sup> *cox2-62*]). For clarity the auxotrophic markers mutations in the nuclear genomes of the strains are omitted, but the complete genotypes of all strains are included in the **Table 1**). To do that the ρ⁺*cox2-62* mtDNA was cytoduced from the NB40-3C strain (*Mata* [ρ⁺*cox2-62*]) (Steele et al., 1996) to the *Matα kar1-1* [ρ⁰] strain
giving the YTMT2 strain (*Matα kar1-1* [ρ⁺ cox2-62]). Then the YTMT2 was crossed to MR10 (*Mata* [ρ⁺ atp6::ARG8m]). After crossing, cells were allowed to divide during 20-40 generations to allow mtDNA recombination and mitotic segregation of the double mutation. The double [ρ⁺ atp6::ARG8m cox2-62] mutant colonies were identified by crossing with two strains: the synthetic ρ⁻ strain SDC30 (Duvezin-Caubet et al., 2003) that carries the synthetic mtDNA containing only *ATP6* and *COX2* genes (*Matα kar1-1* [ρ⁻ COX2 *ATP6*]) and the YTMT2 strain (*Matα kar1-1* [ρ⁺ cox2-62]). The RKY83 clones producing the respiratory competent progeny with SDC30 but not with the YTMT2 were bearing the mtDNA [ρ⁺ atp6::ARG8m cox2-62]. This strain was the recipient one of the *ATP6* gene flanked by the regulatory sequences of the *COX2* gene. In the second step the donor strain of the [ρ⁻ 5'UTR<sub>COX2</sub> *ATP6* 3'UTR<sub>COX2</sub>] was constructed. The *ATP6* gene flanked by 75 bp of 5'UTR and 118 bp of 3'UTR of *COX2* was synthesized using GeneArt technology (ThermoFisher Scientific) and cloned at the *EcoRI* site of pJM2 plasmid containing already the *COX2* coding sequence (Thorsness & Fox, 1993), giving pRK49-2. This plasmid was introduced by the biolistic bombardment into the DFS160 strains (*Matα kar1-1* [ρ⁰]) giving the synthetic ρ⁻ strain RKY89 (*Matα kar1-1* [ρ⁻ 5'UTR<sub>COX2</sub> *ATP6* 3'UTR<sub>COX2</sub> *COX2*]). The purification of the synthetic ρ⁻ mitochondrial transformants after the biolistic transformation by sub-cloning and re-crossing with the NB40-3C is shown and described in the legend to **Figure 3** [*Insert Figure 3 here*]. The third step was the cross of RKY89 with RKY83, which resulted in the respiratory competent progenies, named RKY112 (*Mata* [ρ⁺ atp6::ARG8m 5'UTR<sub>COX2</sub> *ATP6* 3'UTR<sub>COX2</sub>], which were growing on minimal medium without arginine. This strain was the recipient one of the 5'UTR<sub>ATP6</sub> *GFP<sub>β1-10</sub>* 3'UTR<sub>ATP6</sub> gene flanked by the regulatory sequences of the *ATP6* gene.

### 1.2.2. Construction of the RKY176 BiG Mito-Split-GFP strain

To integrate *GFP<sub>β1-10</sub>* gene into mtDNA its sequence first was cloned into the pJM2 plasmid. The DNA fragment encoding first ten beta-sheets of *GFP* flanked by the regulatory sequences of *ATP6* gene and *BamHI-EcoRI* restriction sites was synthesized by GenArt technology. The sequence of this DNA is published in the reference (Bader et al., 2020). The *BamHI-EcoRI* DNA fragment was cloned into pJM2 plasmid giving the pRK67-2, which was introduced into mitochondria of DFS160 strain by biolistic transformation. The synthetic ρ⁻ strain RKY172 (*Matα kar1-1* [ρ⁻ 5'UTR<sub>ATP6</sub>
GFP<sub>β1-10</sub> 3'UTR<sub>ATP6</sub> COX2]) was purified as above (Figure 3) and crossed with strain RKY112. This cross resulted in the respiratory competent progenies, named RKY176 (Mata [ρ<sup>+</sup> atp6::GFP<sub>β1-10</sub> 5'UTR<sub>COX2</sub>ATP63'UTR<sub>COX2</sub>], which were not able to grow on minimal medium without arginine, with GFP<sub>β1-10</sub> gene sequence integrated in the locus of ATP6 under the regulatory sequences of ATP6 gene (Bader et al., 2020).

### 1.3. β11-chapelet tagging of nuclear-encoded proteins

We engineered a Gateway destination plasmid named pAG414-pGPD-ccdB-β11ch plasmid by inserting the β11ch coding sequence (Figure 4) ([Insert Figure 4 here]), that was synthesized by Genscript, into the pAG414-pGPD-ccdB Gateway destination vector (Alberti et al., 2007) as described (Bader et al., 2020). In most cases, we are not taking advantage of the Gateway system to distribute the gene of interest in various destination vector and we setup an isothermal assembly approach to tag the genes with the GFP<sub>β11ch</sub> marker. During this time course of these experiments, we removed the Gateway features from the GFP<sub>β11ch</sub> plasmids. Here we describe only the isothermal assembly approach (Gibson et al., 2009) but we can still provide our original destination vector.

### 1.4. Overview of the use of Big Mito-Split-GFP for probing mitochondrial translocation of a protein

The gene of interest (GOI) that has been GFP<sub>β11ch</sub>-tagged can be expressed using different strategies in the BiG Mito-Split-GFP strain. The quickest and easiest approach is to express it from Yeast Centromere plasmids (YCp, low copy vectors) as described in section 1.3. This approach is also the one we recommend for expression of GFP<sub>β11ch</sub>-tagged genes which are not from S. cerevisiae. Note that in this case, if a yeast ortholog exists, we recommend cloning of the GOI under the dependence of the promoter and terminator of the yeast ortholog. Along the same line, for yeast GOI chromosomal integration of GFP<sub>β11ch</sub> sequence at the 3'-end of the GOI using the CRISPR/Cas9-based method or homologous recombination, can be made in order to preserve the endogenous promoter and keep physiological levels of expression of the GFP<sub>β11ch</sub>-tagged GOI. On the opposite, it's also possible to overexpress a GFP<sub>β11ch</sub>-tagged protein by cloning the GFP<sub>β11ch</sub>-tagged GOI in a Yeast Episomal high copy plasmid (YEp). Furthermore, while optimal growth conditions are described in section 3.3., liquid culture media can be supplemented by any stress molecules that would be
required if mitochondrial relocation is supposed to be stress-induced. The main
advantage with the BiG Mito-Split-GFP system, is to be capable of only visualizing the
GFP signal of the mitochondrial echoform of the dual-localized GFP<sub>β11ch</sub>-tagged protein
by any type of fluorescence microscope. [Insert Figure 5 here]. **Figure 5** shows that
the mitochondrial network can already be easily detected with an epifluorescence
microscope, but using a confocal and high-resolution microscope yield high quality
images. Note that when transformation of the BiG Mito-Split-GFP with a plasmid-borne
GFP<sub>β11ch</sub>-tagged GOI does not yield any mitochondrial GFP signal, expression of
GFP<sub>β11ch</sub>-tagged protein and verification that the BiG Mito-Split-GFP strain did not lose
the edited mitochondrial DNA, by immunodetection using antibodies recognizing
specifically GFP<sub>β11ch</sub> and GFP<sub>β1-10</sub> (see section 3.4 and **Table 2**) is mandatory prior
concluding that the protein encoded by the GOI is not mitochondrial or does not yield
a mitochondrial echoform.

**2. Materials**

**2.1. For cloning and engineering of strains**

- Thermal cycler

- 5× stock mix : 3 mL 1 M Tris-HCl pH 7.5, 150 µL 2 M MgCl₂, 600 µL of dNTP mix (10 mM each), 300 µL 1 M DTT, 300 µL 100 mM NAD+, 1.5 g of PEG-8000 and sterile ultrapure water up to 6 mL final volume. Store 320 µL aliquots in a -80 °C freezer. This mix can be kept for several years.

- 2× reaction mix : 320 µL 5´ isoT master mix, 1.2 µL of T5 exonuclease (New England Biolabs, 10 U/µL), 20 µL Phusion DNA polymerase (ThermoFisher Scientific, 2 U/µL), 8 µL Taq DNA ligase (New England Biolabs, 40 U/µL) and water up to 800 µL. Freeze 11 µL aliquots and keep at -80 °C.

- Dry Heating Block for 1,5 mL microtubes

- Competent *E.coli* cells : We usually use XL1 blue cells prepared as described (Inoue et al., 1990).

- BiG Mito-Split-GFP Strain (see **Table 1**).

- Liquid Yeast extract Peptone Dextrose (YPD): Peptone 2 % (w/v), Yeast extract 1 % (w/v), Dextrose 2 % (w/v). Autoclaved at 120 °C, 1.2 bars for 20 min.

- Bench Top Centrifuge.

- Dry Heating Block for 1.5 mL microtubes.
- Salmon sperm DNA (10 mg/mL).

- Transformation buffer 1×: polyethylene glycol PEG-4000 40% (w/v), 0,1 M lithium acetate, 10 mM TrisHCl pH 7,5, 1 mM Na2EDTA

- Water bath.

- Solid Synthetic Complete Dextrose without tryptophane (SCD-Trp): Yeast Nitrogen Base (without amino acids) 0.675 % (w/v), dextrose 2 % (w/v), amino acid (-Trp) dropout mix, agar 2 % (w/v). Autoclaved at 120 °C, 1.2 bars for 20 min.

- Glass beads (2.5 - 4.5 mm in diameter).

- 30 °C incubator.

## 2.2. For cell growth

- Liquid Synthetic Complete Galactose without tryptophane (SCGal-Trp): Yeast Nitrogen Base (without amino acids) 0.675 % (w/v), galactose 2 % (w/v), amino acid (-Trp) dropout mix. Do not autoclave, but rather use vacuum filtration system with 0.2 µm pores.

- 30 °C incubator Shaker

## 2.3. For protein immunodetection

- 1,85 M NaOH

- Trichloroacetic acid 100 % (w/v).

- Laemmli buffer 1×: 0.5 M Tris-HCl pH 6.8, SDS 8 % (w/v), glycerol 40 % (v/v), Bromophenol blue 0.05 % (w/v)

- Mini-PROTEAN®3 System Glass Plates (BIO-RAD)

- SDS polyacrylamide gels. The resolving gel is composed of acrylamide:bisacrylamide (30 % 37.5:1) supplemented with 0.5 % (v/v) 2,2,2-Trichloroethanol (TCE) (optional, see section 3.4.) diluted in 450 mM Tris-HCl pH 8.8, SDS 0.12 % (w/v) and the acrylamide concentration is adjusted depending on the molecular weight of the proteins that will be analyzed (usually 8-, 10- or 12 % (v/v)). After addition of ammonium persulfate (APS) 0.1 % (w/v) and N,N,N',N Tetramethylethylenediamine (TEMED) 0.1 % (v/v), immediately pour the solution between two 10 × 8 cm Mini-PROTEAN® Spacer Plates from BIO-RAD with integrated spacers of 0.75-, 1- or 1.5 mm. Cover the surface with ethanol 100 % and discard the ethanol after polymerization. The stacking gel, which is poured on top of the resolving gel, is composed of 5 % (v/v)
acrylamide:bisacrylamide (30 % 37.5:1) supplemented with TCE diluted in 125 mM Tris-HCl pH 6.8, SDS 0.1 % (w/v). The polymerization is initiated by addition of APS 0.1 % (w/v) and TEMED 0.1 % (v/v) and the comb is added right after pouring the gel.

- Mini-PROTEAN® Tetra Vertical Electrophoresis Cell from BIO-RAD

- PowerPac™ Basic Power Supply

- Mini Trans-Blot® Cell from BIO-RAD

- Blue cooling unit

- Gel holder cassettes

- Foam pads

- 7,5×9 cm Whatman™ papers

- 7,5×9 cm Whatman™ Protran® BA85 nitrocellulose western blotting membranes

- Transfer buffer 1× : 3 g/L Tris, 14,4 g/L Glycine, 0,1 g/L SDS, 20 % (v/v) ethanol.

- PowerPac™ HC Power Supply

- TBS-Tween20: 50 mM Tris-HCl pH 7.6, 150 mM NaCl, and Tween20 0.1 % (v/v).

- Skim milk powder

- Heidolph Duomax shaker

- Primary and secondary antibodies (see Tables 2 and 3)

- Clarity Western ECL substrate from BIO-RAD

- Chemidoc™ imaging system from BIO-RAD

- Red Ponceau

## 2.4. For fluorescence microscopy

- Liquid SC-Gal: 0.675 % (w/v) Yeast Nitrogen Base (without amino acids), 2 % (w/v) galactose, amino acid (-Trp) dropout mix. Do not autoclave, but rather use vacuum filtration system with 0.2 µm pores.

- MitoTracker™ Red CMXRos (Thermo Fisher)

- 7,6×2,6 cm microscope slides and 2,2×2,2 cm cover slips

- Zeiss LSM 800 confocal microscope with a 63× apochromatic plane objective (1.40 digital aperture)

- Zeiss AxioObserverD1 epifluorescence microscope with an apochromatic 100× (1.45 digital aperture)

# 3. Methods
### 3.1. GFP<sub>B11ch</sub> epitope tagging

- PCR amplify the coding sequence of your gene of interest (GOI) using I1-for and I2-rev primers. Notice that primer extensions for isothermal assembly usually are 20-25 nucleotides long. Do not forget to remove the stop codon. You might purify the PCR product using the Macherey Nagel PCR cleanup kit on a PCR product that has been run or not on an agarose gel. Usually, we do not purify the PCR products when having a single PCR band.

- To obtain the plasmid into which the PCR product will be cloned by isothermal assembly, we usually PCR-amplify the plasmid in two parts that will be separated within the ampicillin resistance gene. Doing so, enhances the proportion of positive clones.

- The assembly of the recombinant plasmid by isothermal assembly is performed as follows : 11 µL 2× reaction mix is added to 9 µL DNA solution that is classically obtained by mixing PCR fragments (1-5 fragments, approximately 1 µL each) and sterile ultrapure water. The reaction mixture is immediately incubated at 50 °C for 30-60 min. Parental plasmid DNA present within PCR products can be eliminated via *DpnI* digestion (e.g. we simply add 2 µL FastDigest restriction buffer 10× and 1 µL FastDigest *DpnI*, incubate the mixture for 30-60 min at 37 °C and heat inactivate *DpnI* at 95 °C for 5 min). 5 µL of the whole mixture is used to transform chemically-competent *E. coli* cells and yields classically 10-800 colonies.

### 3.2. Transformation of the BiG Mito-Split-GFP strain

Transformations are carried out on yeast cells in exponential growth phase.

- The day before transformation, launch an overnight (ON) culture in 2 mL liquid YPD medium.

- Dilute in the morning in 20 mL of the same medium at OD<sub>600nm</sub> = 0.2 and incubate under a 200 rpm agitation at 30 °C until reaching an OD<sub>600nm</sub> of 0.5 to 0.8 (4-5 h of growth).

- Take a volume corresponding to 10 units of OD<sub>600nm</sub> and centrifuge at 6 000 × g for 1 min at room temperature (RT).

- Wash the pellet with 10 mL of milliQ water and resuspend the pellet with 10 µL of salmon sperm DNA (0,2 µg/mL final) previously denatured at 95 °C for 10 min and 1 µg of plasmid DNA. Add 500 µL of transformation buffer and incubate 15 min at RT with inversion mixing every 5 min.

- Apply a thermal shock of 15 min in a water bath heated at 42 °C.
- Centrifuge cells at 6 000 × g for 1 min and wash the pellet with 1mL milliQ water

- Recover the pellet and resuspend it in 100 µL milliQ water to spread it on a Petri dish containing solid SCD-Trp medium using sterile glass beads.

- Incubate the plate at 30 °C for 2 to 3 days until clones appear.

- Streak 4-6 colonies on a new SCD-Trp plate and incubate at 30 °C for 1 to 2 days.

### 3.3. Growth of the BiG Mito-Split-GFP strain expressing GFP<sub>β11ch</sub>-tagged proteins

- Launch ON precultures of 3-4 selected clones in 2 mL liquid SC Galactose (SCGal)-Trp medium.

- Dilute the next day in the same medium at OD<sub>600nm</sub> = 0.2: for western-blot experiments dilute in 10 mL and in 2 mL for microscopy.

- Incubate under a 200 rpm agitation at 30 °C until an OD<sub>600nm</sub> of 0.5 to 0.8 is achieved (usually, it takes 4 to 5 hours).

- Once the exponential phase is reached, use this culture for protein extractions and microscopy imaging as described in sections 3.4 and 3.5.

### 3.4. Protein extraction and Western-blot analyses

To check if BiG Mito-Split-GFP transformation is successful, you can proceed with a Western-blot to confirm the GFP<sub>β11ch</sub>-tagged protein expression.

- Take a volume corresponding to 3 OD<sub>600nm</sub> units of yeast cells prepared in section 3.2. and add 50 µL of YPD medium to help cells precipitation.

- Centrifuge cells at 6 000 × g for 5 min at RT and resuspend the pellet in 500 µL of milliQ water.

- Add 50 µL of 1,85 M NaOH and incubate on ice for 10 min.

- Add 50 µL of TCA 100 % (w/v) and incubate on ice for 10 min.

- Centrifuge at 12 000 × g for 5 min at 4 °C, remove the supernatant, and centrifuge again to remove any remaining supernatant.

- Resuspend pellets in 50 µL of SBR Tris 1× buffer.

- Denature your protein extracts at 42 °C for 10 min. and load 8 µL on 10 % polyacrylamide-SDS (SDS_PAGE) gels. Note that the acrylamide bottle can be supplemented with trichloroethanol (TCE) to directly detect your proteins after the migration using the Chemidoc™ Touch (BIO-RAD) Stain-free system. Migrate in TG-SDS at 200 V until the migration front is below the gel.
- Wet-transfer on nitrocellulose membranes (Protan BA85 Whatman) at 100 V for 90 min and check the transfer efficiency by staining the membrane with red ponceau.

- Block the membrane by a 30 min incubation with TBS Milk 5 % (w/v) at RT.

- Incubate with the primary antibody (diluted in TBS Milk 5 % (w/v) at the appropriate dilution) for 2 h at RT or ON at 4 °C.

- Wash 3 times the membrane with TBS-Tween 0,1 % (v/v) and incubate with the secondary antibody (diluted in TBS Milk 5 % (w/v) at the appropriate dilution) for 1 h at RT.

- Wash 3 times the membrane with TBS-Tween 0,1 % (v/v) before revealing it with an ECL solution. Images are acquired with the Chemidoc™ Touch (BIO-RAD) and primary and secondary antibodies are listed in the **Table 2** and **3** respectively.

**3.5. Visualizing mitochondrial translocation by fluorescence microscopy**

Once clones are validated by Western-blots, new cultures from the same tested clones can be launched to visualize by microscopy the potential mitochondrial import of your protein of interest. To confirm a colocalization with mitochondria, we use the MitoTracker™ Red CMXRos (Thermo Fisher) to stain the mitochondrial network and use a DsRED filter for detection of the fluorescent signal.

- Take a volume of yeast cells in exponential growth phase corresponding to 1 unit of OD<sub>600nm</sub> and add MitoTracker™ Red CMXRos (Thermo Fisher) to a final concentration of 100 µM and incubate the cells in a water bath heated at 42 °C for 15 min.

- Centrifuge cells at 6 000 × g for 5 min at RT and wash the pellet 3 times with 1 mL of milliQ water.

- After the final wash, resuspend cells in 50 µL of SCGal-Trp medium.

- Drop 2,5 µL of stained live yeast cells between microscope slide (7,6×2,6 cm) and cover slip (2×2 cm) for microscopy observation.

- First observations (**Figure 5**) are made on a Zeiss AxioObserverD1 epifluorescence microscope with an apochromatic 100× (1.45 digital aperture) oil immersion objective. GFP and DsRED filter cubes are used for fluorescence observation and DIC (Differential Interference Contrast) for light field observation. The images are captured by a CoolSNAP-HQ2 (Roper Scientific) cooled monochrome camera using the AxioVision (Zeiss) software.
- For better resolution, new images can be took using a confocal microscope. For this review, we used the Zeiss LSM 800 of the imaging platform of the Research Center in Biomedicine of Strasbourg (PIC-STRA, CRBS). A 63× apochromatic plane objective (1.40 digital aperture) with oil immersion, LED illumination (Colibri, Zeiss) and GFP and DsRED filter cubes are used for direct observation using eyepieces. In confocal mode, 488 nm and 640 nm lasers replace the LEDs and high sensitivity photomultipliers (GaAsP, Phospho Arsenide de Gallium) detect the emitted fluorescence. The images are acquired using the Zeiss Zen Blue software, with a laser residence time per pixel (pixel dwell time) of 11.8 µs. They are then analyzed on FIJI (Schindelin et al., 2012) and the figures are produced thanks to the FigureJ plugin (Mutterer & Zinck, 2013).

## Acknowledgements

This work was supported by the Integrative Molecular and Cellular Biology (IMCBio), as part of the Interdisciplinary Thematic Institutes (ITI) 2021-to-2028 program of the University of Strasbourg, CNRS, and INSERM, supported by IdEx Unistra (ANR-10-IDEX-0002) and EUR IMCBio (ANR-17-EURE-0023) under the framework of the French Investments for the Future Program (to S.Z, H.D.B, B.S, S.F); by the University of Strasbourg (to S.Z., H.D.B, B.S, S.F), by the CNRS (to S.Z, H.D.B, B.S, S.F) and the National Science Center of Poland grant nr UMO-2018-31-B-NZ3-01117 (to R.K).

## References

Alberti, S., Gitler, A. D., & Lindquist, S. (2007). A suite of Gateway® cloning vectors for high-throughput genetic analysis in *Saccharomyces cerevisiae*. *Yeast*, 24(10), 913-919. https://doi.org/10.1002/yea.1502

Bader, G., Enkler, L., Araiso, Y., Hemmerle, M., Binko, K., Baranowska, E., De Craene, J.-O., Ruer-Laventie, J., Pieters, J., Tribouillard-Tanvier, D., Senger, B., Di Rago, J.-P., Friant, S., Kucharczyk, R., & Becker, H. D. (2020). Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP. *eLife*, 9, e56649. https://doi.org/10.7554/eLife.56649

Baker, Z. N., Forny, P., & Pagliarini, D. J. (2024). Mitochondrial proteome research: The road ahead. *Nature Reviews Molecular Cell Biology*, 25(1), 65-82. https://doi.org/10.1038/s41580-023-00650-7
Ben-Menachem, R., Tal, M., Shadur, T., & Pines, O. (2011). A third of the yeast mitochondrial proteome is dual localized: A question of evolution. *PROTEOMICS*, 11(23), 4468-4476.
https://doi.org/10.1002/pmic.201100199

Bonnefoy, N., & Fox, T. D. (2002). Genetic Transformation of Saccharomyces cerevisiae Mitochondria. *Methods in Enzymology*, 350, 97-111.

Conde, J., & Fink, G. R. (1976). A mutant of Saccharomyces cerevisiae defective for nuclear fusion. *Proceedings of the National Academy of Sciences*, 73(10), 3651-3655. https://doi.org/10.1073/pnas.73.10.3651

Duvezin-Caubet, S., Caron, M., Giraud, M.-F., Velours, J., & Di Rago, J.-P. (2003). The two rotor components of yeast mitochondrial ATP synthase are mechanically coupled by subunit δ. *Proceedings of the National Academy of Sciences*, 100(23), 13235-13240. https://doi.org/10.1073/pnas.2135169100

Eisenberg-Bord, M., & Schuldiner, M. (2017). Ground control to major TOM: Mitochondria–nucleus communication. *The FEBS Journal*, 284(2), 196-210.
https://doi.org/10.1111/febs.13778

Foury, F., Roganti, T., Lecrenier, N., & Purnelle, B. (1998). The complete sequence of the mitochondrial genome of *Saccharomyces cerevisiae*. *FEBS Letters*, 440(3), 325-331. https://doi.org/10.1016/S0014-5793(98)01467-7

Frechin, M., Enkler, L., Tetaud, E., Laporte, D., Senger, B., Blancard, C., Hammann, P., Bader, G., Clauder-Münster, S., Steinmetz, L. M., Martin, R. P., di Rago, J.-P., & Becker, H. D. (2014). Expression of Nuclear and Mitochondrial Genes Encoding ATP Synthase Is Synchronized by Disassembly of a Multisynthetase Complex. *Molecular Cell*, 56(6), 763-776.
https://doi.org/10.1016/j.molcel.2014.10.015

Frechin, M., Senger, B., Brayé, M., Kern, D., Martin, R. P., & Becker, H. D. (2009). Yeast mitochondrial Gln-tRNA Gln is generated by a GatFAB-mediated transamidation pathway involving Arc1p-controlled subcellular sorting of cytosolic GluRS. *Genes & Development*, 23(9), 1119-1130.
https://doi.org/10.1101/gad.518109

Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred
kilobases. *Nature Methods*, 6(5), 343-345.
https://doi.org/10.1038/nmeth.1318

Inoue, H., Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids. *Gene*, 96(1), 23-28.
https://doi.org/10.1016/0378-1119(90)90336-P

Morgenstern, M., Stiller, S. B., Lübbert, P., Peikert, C. D., Dannenmaier, S., Drepper, F., Weill, U., Höß, P., Feuerstein, R., Gebert, M., Bohnert, M., Van Der Laan, M., Schuldiner, M., Schütze, C., Oeljeklaus, S., Pfanner, N., Wiedemann, N., & Warscheid, B. (2017). Definition of a High-Confidence Mitochondrial Proteome at Quantitative Scale. *Cell Reports*, 19(13), 2836-2852. https://doi.org/10.1016/j.celrep.2017.06.014

Murley, A., & Nunnari, J. (2016). The Emerging Network of Mitochondria-Organelle Contacts. *Molecular Cell*, 61(5), 648-653.
https://doi.org/10.1016/j.molcel.2016.01.031

Mutterer, J., & Zinck, E. (2013). Quick-and-clean article figures with FigureJ. *Journal of Microscopy*, 252(1), 89-91. https://doi.org/10.1111/jmi.12069

Rak, M., Tetaud, E., Godard, F., Sagot, I., Salin, B., Duvezin-Caubet, S., Slonimski, P. P., Rytka, J., & Di Rago, J.-P. (2007). Yeast Cells Lacking the Mitochondrial Gene Encoding the ATP Synthase Subunit 6 Exhibit a Selective Loss of Complex IV and Unusual Mitochondrial Morphology. *Journal of Biological Chemistry*, 282(15), 10853-10864.
https://doi.org/10.1074/jbc.M608692200

Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.-Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P., & Cardona, A. (2012). Fiji: An open-source platform for biological-image analysis. *Nature Methods*, 9(7), 676-682. https://doi.org/10.1038/nmeth.2019

Smalheiser, N. R. (1996). Proteins in unexpected locations. *Molecular Biology of the Cell*, 7(7), 1003-1014. https://doi.org/10.1091/mbc.7.7.1003

Steele, D. F., Butler, C. A., & Fox, T. D. (1996). Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation. *Proceedings of the National Academy of Sciences*, 93(11), 5253-5257. https://doi.org/10.1073/pnas.93.11.5253
Thorsness, P. E., & Fox, T. D. (1993). Nuclear mutations in Saccharomyces cerevisiae that affect the escape of DNA from mitochondria to the nucleus. *Genetics*, **134**(1), 21-28. https://doi.org/10.1093/genetics/134.1.21

Vögtle, F.-N., Burkhart, J. M., Gonczarowska-Jorge, H., Küçükköse, C., Taskin, A. A., Kopczynski, D., Ahrends, R., Mossmann, D., Sickmann, A., Zahedi, R. P., & Meisinger, C. (2017). Landscape of submitochondrial protein distribution. *Nature Communications*, **8**(1), 290.
https://doi.org/10.1038/s41467-017-00359-0

Yogev, O., & Pines, O. (2011). Dual targeting of mitochondrial proteins: Mechanism, regulation and function. *Biochimica et Biophysica Acta (BBA) - Biomembranes*, **1808**(3), 1012-1020.
https://doi.org/10.1016/j.bbamem.2010.07.004
# Tables

<table><caption>**Table 1**
Genotypes and origins of yeast strains</caption><thead><tr><td>Strain</td><td>Nuclear genotype</td><td>mtDNA</td><td>Source</td></tr></thead><tbody><tr><td>MR6</td><td>MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3</td><td>ρ<sup>+</sup></td><td>Rak et al., 2007a</td></tr><tr><td>DFS160</td><td>MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1</td><td>ρ<sup>o</sup></td><td>Steele et al., 1996</td></tr><tr><td>NB40-3C</td><td>MATa lys2 leu2-3,112 ura3-52 his3ΔHindIII arg8::hisG</td><td>ρ<sup>+</sup> cox2-62</td><td>Steele et al., 1996</td></tr><tr><td>MR10</td><td>MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::hisG</td><td>ρ<sup>+</sup> atp6::ARG8<sup>m</sup></td><td>Rak et al., 2007a</td></tr><tr><td>SDC30</td><td>MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1</td><td>ρ-COX2 ATP6</td><td>Rak et al., 2007b</td></tr><tr><td>YTMT2</td><td>MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1</td><td>ρ+cox2-62</td><td>Kabala et al., 2022; Bader et al., 2020</td></tr><tr><td>RKY83</td><td>MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 arg8::HIS3</td><td>ρ+cox2-62 atp6::ARG8<sup>m</sup></td><td>Kabala et al., 2022; Bader et al., 2020</td></tr><tr><td>RKY89</td><td>MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1</td><td>ρ- 5'UTR<sub>COX2</sub> ATP6 3'UTR<sub>COX2</sub> COX2</td><td>Kabala et al., 2022; Bader et al., 2020</td></tr><tr><td>RKY112</td><td>MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 arg8::HIS3</td><td>ρ+ atp6::ARG8<sup>m</sup> 5'UTR<sub>COX2</sub> ATP6 3'UTR<sub>COX2</sub></td><td>Kabala et al., 2022; Bader et al., 2020</td></tr><tr><td>RKY172</td><td>MATα leu2Δ ura3-52 ade2-101 arg8::URA3 kar1-1</td><td>ρ- 5'UTR<sub>ATP6</sub> GFP<sub>β1-10</sub> 3'UTR<sub>ATP6</sub> COX2</td><td>Bader et al., 2020</td></tr><tr><td>RKY176</td><td>MATa ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3</td><td>ρ+ atp6::GFP<sub>β1-10</sub> 5'UTR<sub>COX2</sub> ATP6 3'UTR<sub>COX2</sub></td><td>Bader et al., 2020</td></tr></tbody></table>
<table><caption>**Table 2**
Primary used in this study</caption><thead><tr><td>Specificity</td><td>Name</td><td>Type</td><td>Vendor</td><td>Dilution</td></tr></thead><tbody><tr><td>GFPβ<sub>1-10</sub></td><td>Anti-GFP N-terminal</td><td>Rabbit polyclonal</td><td>Sigma (#G1544)</td><td>1:5000</td></tr><tr><td>GFPβ<sub>11ch</sub></td><td>Anti-GFP</td><td>Mouse monoclonal IgG1κ clones 7.1 and 13.1</td><td>Roche (11814460001)</td><td>1:5000</td></tr></tbody></table>

<table><caption>**Table 3**
Secondary antibodies used in this study</caption><thead><tr><td>Targeted primary antibody</td><td>Type</td><td>Dilution</td></tr></thead><tbody><tr><td>Anti-GFP N-terminal</td><td>Goat-Anti-rabbit-HRP</td><td>1:5000</td></tr><tr><td>Anti-GFP</td><td>Goat-Anti-mouse-HRP</td><td>1:5000</td></tr></tbody></table>
## Figure legends

**Figure 1.** Figure 1 title: Comparison between the visualization of a dual-localized protein fused to a regular GFP and expressed in a conventional strain versus the same dual-localized protein tagged with GFP<sub>β11</sub> and expressed in the BiG Mito-Split-GFP strain. – Figure 1 caption: Because GFP<sub>β1-10</sub> and GFP<sub>β11</sub> are translated in separate compartments, only GFP<sub>β11</sub>-tagged mitochondrial proteins or mitochondrial echoforms of GFP<sub>β11</sub>-tagged dual localized proteins (cytosolic and mitochondrial) will trigger GFP reconstitution upon self-assembly of GFP<sub>β1-10</sub> with GFP<sub>β11</sub> and can be visualized by fluorescence microscopy of living cells.

**Figure 2.** Figure 2 title: A schematic representation of the steps and intermediate strains necessary to construct the BiG Mito-Split-GFP strain in which the mtDNA was edited to express the GFP<sub>β1-10</sub> fragment of a split-GFP: RKY83 (A), RKY112 (B) and RKY176 (C). – No caption for Figure 3.

**Figure 3.** Figure 3 title: Engineering of the RKY89 strain by biolistic transformation of the pKR49 plasmid. – Figure 2 caption: Shooting of 10<sup>9</sup> cells of the ρ<sup>0</sup> strain DFS160 plated on biolistic medium lacking leucine with plasmids Yep351 containing the *LEU2* gene and pRK49 results in the emergence of nuclear Leu<sup>+</sup> transformants of which some contain pRK49 in their mitochondria (ρ<sup>-</sup> RKY89). The Leu<sup>+</sup> transformants containing pRK49 in their mitochondria are identified by their ability to produce Gly<sup>+</sup> clones after crossing with a layer of NB40-3C cells [ρ<sup>+</sup> cox2-62], plated on rich glucose medium, due to recombination between the COX2 gene present in pRK49 (therefore the synthetic ρ<sup>-</sup> transformant) and a partial deletion of COX2 (cox2-62) in NB40-3C. A ρ<sup>-</sup> RKY89 clone is picked up from a duplicate on minimal-Leu of the
shoot plate and re-crossed with NB40-3C for genotype confirmation. Usually this step needs to be repeated a few times.

**Figure 4.** [Figure 4 title: *GFP<sub>β11ch</sub> tagging at the 3'-end of a gene of interest using the isothermal assembly strategy*. – Figure 4 caption: (A) Sequence (Nucleotides and amino acids) of the *GFP<sub>β11ch</sub> tag. The β-strands of the *GFP<sub>β11ch</sub> tag* are in green and the spacers between the *GFP<sub>β11</sub>* fragments are in black. Nucleotides in pink correspond to the downstream isoT tag (used in V1 and G1 primers). Note that underlined nucleotides show *EcoRI* and *XhoI* restriction sites at the beginning and at the end of the *GFP<sub>β11ch</sub>* respectively. (B) In silico assembly of the desired construct (p414-pGPD-GOI-GFP<sub>β11ch</sub>) showing the bacterial selection marker (*Amp*<sup>R</sup> (ampicillin resistance), yellow), the gene of interest (GOI, blue), the *GFP<sub>β11ch</sub>* tag (green) with a small linker region (pink), the GPD promoter (pGPD, khaki) and its small linker region (grey). Other elements of the plasmid e.g. the yeast auxotrophy marker *TRP1*, the *E. coli* origin of replication (Co/E1) and the yeast centromeric origin of replication (ARS/CEN) are just indicated on the plasmid map.

**Figure 5.** [Figure 5 title: *Visualization, by fluorescence microscopy, of the reconstituted GFP signal yielded by GFP<sub>β-11ch</sub>-tagged mono- or dual-localized proteins expressed in the BiG Mito-Spli-GFP strain*. – Figure 5 caption: (A) schematic representation adapted from Bader et al., 2020 of the subcellular localization of the *GFP<sub>β-11ch</sub>-tagged proteins* visualized in (B). (B) Visualization of the Split-GFP reconstitution signal using the Zeiss LSM 800 confocal microscope with a 63 × apochromatic plane objective (1.40 digital aperture) (left panel) and the Zeiss AxioObserverD1 epifluorescence microscope with an apochromatic 100× (1.45 digital
aperture) (right panel); scale bar: 5 µm. Mitochondria were labeled using Mito tracker™
CMXRos (left panel).
<figure><img src="image_2.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_5.png" /></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>
A GFP<sub>β11ch</sub> sequence

ggc tgc agg <u>aat</u> tcg ata tca agc tta CGT GCA CAA GCT AGC GGC GGA TCA ACA AGT AGA
G C R N S I S S L R A Q A S G G S T S R

GAT CAT ATG GTT TTG CAC GAA TAT GTC AAT GCT GCT GGT ATT ACT GGT ACC GGT GGT GGT
D H M V L H E Y V N A A G I T G T G G G

TCT GGT GGT GGT TCT ACC AGT AGA GAT CAT ATG GTT TTG CAC GAA TAT GTC AAT GCT GCT
S G G G S T S R D H M V L H E Y V N A A

GGT ATT ACT GGT ACA GGT GGT GGT TCT GGT GGT GGT TCT ACC AGT AGA GAT CAT ATG GTT
G I T G T G G G S G G G S T S R D H M V

TTG CAC GAA TAT GTC AAT GCT GCT GGT ATT ACT GGT ACC TGA <u>ctc</u> gag
L H E Y V N A A G I T G T *

B GFP<sub>β11ch</sub> plasmid assembly

<figure><img src="image_10.png" /></figure>

Primers

A1 : TTTTCTGTGACTGGTGAGTACTCAACC

A2 : TTGAGTACTCACCAGTCACAGAAAAGC

V1 : GGCTGCAGGAATTCGATATCAAGC

V2 : TAGTTCTAGAATCCGTCGAAAC

G1 : GCTTGATATCGAATTCCTGCAGCC- 20-25 nt of GOI
without stop codon

G2 : GTTTCGACGGATTCTAGAACTA - ATG of GOI+20-25 nt

PCR products

Plasmid : A1/V1 and A2/V2

GOI : G1/G2
<figure><img src="image_11.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>