Plasmid Construction and Fluorescent Protein Tagging

All plasmids were constructed using the pEGFP-C1 or -N1 backbone (Takara Bio Inc.). When different fluors were used, EGFP was replaced with CFP, mTq2 (mTurquoise2; a gift from T. Gadella, University of Amsterdam, Amsterdam, Netherlands; Goedhart et al., 2012 (link)), mRFP (Campbell et al., 2002 (link)), mCherry (Shaner et al., 2004 (link)), or iRFP (Filonov et al., 2011 (link)).
The following cDNAs were amplified by PCR and subcloned into plasmids as follows: residues 546–647 from L. pneumophila SidM (available from GenBank under accession no. DQ845395), corresponding to the isolated P4M domain (Schoebel et al., 2010 (link)), were cloned into pEGFP-C1 (or spectral variants mCherry or iRFP) at BspEI and EcoRI sites. We also made a similar plasmid inserting the same P4M fragment at SalI and BamHI sites with a GGSASGLRS linker between GFP and the N terminus. This was used as a template to insert a second P4M insert at EcoRI and SalI sites with the same linker between GFP and the first P4M and a GGSAVDGGSASGLRS linker separating the tandem P4M domains. To generate FRB fused to canine Rab5 and Rab7 (a gift from R. Lodge, Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada; Rojas et al., 2008 (link)), the entire coding regions were inserted at HindIII–KpnI sites of a modified piRFP-C1 vector containing the FRB domain (NCBI Nucleotide accession no. NM_004958; residues 2,021–2,113) flanked by GGAGA and GGSAGGSA linkers at the 5′ and 3′ ends, respectively, and inserted at BglII–HindIII sites. For pmCherry-C1-FKBP-MTM1, FKBP (NCBI Nucleotide accession no. NM_054014; residues 3–109) flanked by GAGGAARAAL and (SAGG)₅PRAQASNSA linkers at the 5′ and 3′ ends were inserted at NotI–SalI sites and MTM1(NCBI Nucleotide accession no. NM_000252) at SalI–BamHI. Hepatitis C NS5A (Budhu et al., 2007 (link)) was obtained from Addgene and inserted at HindIII–KpnI sites in pmCherry-N1. piRFP-N1-TTC7B (isolated from an EST that misses L53-A85 from exon 2; GenBank accession no. BQ426031) was inserted at NheI–SalI sites with a C-terminal GGSAGGSA linker with the iRFP. Constructs are available through Addgene. PI4KAv1 (NCBI Nucleotide accession no. NM_058004) was inserted at EcoRI–SalI sites of pmTq2-C1, and PI4KBv2 (NCBI Nucleotide accession no. NM_001198773) was inserted at XhoI–KpnI sites in pEGFP-C1. pEGFP-N1-EFR3B (NCBI Nucleotide accession no. NM_014971) was inserted at NheI–AgeI sites. Murine Sacm1l (NCBI Nucleotide accession no. NM_030692) was inserted at BglII–SalI sites in pmCherry-N1. Additional plasmids were obtained as follows: pEGFP-N1-PH-PLCδ1 (Várnai and Balla, 1998 (link)); pEGFP-N1-PH-FAPP1 and pEGFP-C1-PH-OSBP (Balla et al., 2005 (link)); PJ, its catalytic mutants, and pECFP-N1-Lyn₁₁-FRB (Hammond et al., 2012 (link)); pmRFP-FKBP-INPP5E (Várnai et al., 2006 (link)); pECFP-FRB-giantin^{3,140–3,269} (a gift from T. Inoue, Johns Hopkins University School of Medicine, Baltimore, MD; Komatsu et al., 2010 (link)) or a pmCherry variant; pECFP-C1-FKBP-PIP5K (Suh et al., 2006 (link)); pmCherry-C1-Rab5 and -Rab7 (Rojas et al., 2008 (link)); GFP-FYVE-EEA1 (Balla et al., 2000 (link)); pEGFP-N1-PI4K2A (Jović et al., 2012 (link)); pEGFP-N1-PI4K2B (Balla et al., 2002 (link)); and piRFP-C1-PH-PLCδ1 (a gift from P. De Camilli, Yale School of Medicine, New Haven, CT; Idevall-Hagren et al., 2012 (link)).