Expression plasmids

All amplifications were performed using the Q5 high fidelity or Phusion high fidelity PCR kits (NEB, Ipswich, MA). Restriction enzymes used for cloning procedures were purchased from New England Biolabs (NEB).

To generate P_nhx-2 CemOrange2, P_nhx-2 CemNeptune2.5, and P_nhx-2 CemCardinal2 constructs (pJR2956, pJR2955 and pJR2953), minigene blocks with three synthetic introns (gblock) containing C. elegans codon optimized version of mOrange2 (CemOrange2), mNeptune2.5 (CemNeptune2.5) and mCardinal2 (CemCardinal2) with additional N- and C-terminal restriction sites were synthesized (IDT, Skokie, IL) and sub-cloned into the NheI/SacI restriction sites of the canonical expression vector, pPD49.26 [17, 19]. A 2kb nhx-2 promoter fragment was amplified (S1 Table, primer set 1) and ligated into the HindIII/XbaI restriction sites to yield the final constructs: pJR2956, pJR2955, and pJR2953, respectively.

To generate expression constructs of cloned fragments the NEBuilder HiFi DNA Assembly Cloning Kit was used (NEB). For N-terminus CemOrange2 fusions, fragments were cloned into the KasI restriction site and for C-terminus protein fusions fragments were cloned into the NheI restriction site of pJR2956. P_nhx-2sqst-1::CemOrange2 (pBT3037), P_nhx-2aqp-1::CemOrange2 (pBT3102), P_nhx-2lmn-1::CemOrange2 (pBT3035), P_nhx-2lmp-1::CemOrange2 (pBT2999), P_nhx-2aman-2::CemOrange2 (pLS2965), and P_nhx-2glo-1::CemOrange2 (pBT3038) were generated by amplifying the sqst-1 (S1 Table, primer set 2), aqp-1 (S1 Table, primer set 3), lmn-1 (S1 Table, primer set 9), lmp-1 (S1 Table, primer set 11), aman-2 (S1 Table, primer set 15), and glo-1 (S1 Table, primer set 16) genomic DNA fragment and ligating the fragment into the NheI site of P_nhx-2 CemOrange2 (pJR2956), respectively. P_nhx-2CemOrange2::lgg-1 (pBT3043), P_nhx-2CemOrange2::cup-5 (pBT3011), P_nhx-2CemOrange2::rab-5 (pBT3007), P_nhx-2CemOrange2::rab-7 (pBT3014), P_nhx-2CemOrange2::tram-1 (pBT3000), and P_nhx-2CemOrange2::pisy-1 (pBT3003) were generated by amplifying the lgg-1 (S1 Table, primer set 4), cup-5 (S1 Table, primer set 8), rab-5 (S1 Table, primer set 12), rab-7 (S1 Table, primer set 13), tram-1 (S1 Table, primer set 14), and pisy-1 (S1 Table, primer set 17) genomic DNA fragment and ligating the fragment into the KasI site of P_nhx-2 CemOrange2, respectively.

Mitochondrial targetting (^mt), peroxisome targeting peptide (SKL) and nuclear localization sequences (NLS) were inserted into the P_nhx-2CemOrange2 expression construct using the Q5 site-directed mutagenesis kit (NEB). P_nhx-2^mtCemOrange2 (pBT3024) was generated using primer set 5 (S1 Table). P_nhx-2NLS^SV-40::CemOrange2::NLS^egl-13 (pBT3047) was generated using primer sets 6 (SV-40) and 7 (egl-13) (S1 Table). P_nhx-2CemOrange2::SKL (pBT3019) was generated using primer set 10 (S1 Table).

To generate P_nhx-2sGFP::KDEL (pOL2184), the nhx-2 promoter was amplified (S1 Table, primer set 1) and sub-cloned into the HindIII/XbaI sites of pPD95.85 to create P_nhx-2sGFP (pAV1771). The stop codon of GFP was mutated by Quikchange site-directed mutagenesis (S1 Table, primer set 18) to the ER retention motif, KDEL, yielding the final construct, pOL2184.

To generate P_nhx-2mKate2 (pBC2370), mKate2 was amplified from pmKate2 (Evrogen, RU) using primer set 21 (S1 Table). A 2kb nhx-2 promoter fragment was amplified (S1 Table, primer set 1) and ligated into the HindIII/XbaI restriction sites of the canonical expression vector, pPD49.26 to yield the final constructs pBC2370. To generate P_nhx-2mKate2::lgg-1 (pME2707-pME2710), additional restrictions sites were added to P_nhx-2mKate2 (S1 Table, primer set 22) and lgg-1 genomic DNA was amplified (S1 Table, primer set 23) and ligated into the PstI/SacI restriction sites to yield the final constructs pME2707-pME2710.

To generate P_nhx-2abt-4::mKate2 (pBT3111), abt-4 genomic DNA was amplified using primer set 19 (S1 Table) and ligated into the NheI site of P_nhx-2mKate2. To generate P_nhx-2abt-4^L162P::mKate2 (pBT3114), pBT3111 was mutated with primer set 20 (S1 Table) using the Q5 site-directed mutagenesis kit.