Fig 1.
Amino terminal hydrophobic residues mediate colocalization of YlfA and YlfB with eukaryotic membrane networks.
(A) Predicted structural motifs of YlfA and YlfB. The amino terminal hydrophobic domains (HD), in grey, were identified by analysis of the YlfA and YlfB amino acid sequences using Kyte-Doolittle [48], TMBase25 [49] and TMHMM2.0 [50] algorithms. Regions predicted to form coiled coil domains (CC), in black, were defined using the COILS server. (B-E) Cellular localization of GFP-tagged Ylf proteins. CHO cells were transfected with plasmids encoding amino-terminal GFP-tagged proteins: full length YlfA (B), YlfA 122–425 (C), full length YlfB (D), and YlfB 121–405 (E). 18 h after transfection, cells were fixed and endogenous KDEL-containing proteins were stained with a mouse anti-KDEL antibody, followed by an AlexaFluor 594-conjugated anti-mouse secondary antibody. Grey scale images of confocal sections show the localization of GFP-tagged proteins and KDEL-containing proteins demarcating the early secretory pathway. Merged color images show GFP-Ylf fusion proteins (green) and anti-KDEL staining (red).
Fig 2.
Amino terminal hydrophobic residues mediate association of YlfA and YlfB with eukaryotic lipid.
HEK293T cells were transfected for 14 h with plasmids encoding full length (A and B) or truncation mutant Ylf proteins (A). A) Membrane-associated and soluble protein fractions were generated by centrifugation of whole cell lysate at 100,000 x g, followed by solubilization of membrane-associated proteins with 0.1% Triton X-100. The presence of Ylf protein in each subcellular fraction is detected by anti-YlfA and anti-YlfB immunoblot. To demonstrate effective fractionation of cell lysates, fractions were probed with antibodies against the transmembrane protein Calnexin and cytosolic alpha-Tubulin. B) Whole cell lysates were overlayed on a 4–26% continuous OptiPrep gradient and centrifuged at 100,000 x g for 3 h. The presence of Ylf protein in each odd fraction was detected by immunoblot using anti-YlfA or anti-YlfB antibodies. The sedimentation profile of membranes associated with the ER and early secretory pathway was revealed by anti-Calnexin immunoblot.
Fig 3.
Co-production of YlfA and YlfB alters localization of YlfB.
CHO cells were co-transfected for 18 h with plasmids encoding amino terminal RFP or GFP-tagged full length YlfA and YlfB. Cells were fixed in 2% PFA, mounted and imaged. Grey scale images show the localization of RFP and GFP-tagged proteins in confocal sections. Merged color images reveal colocalization of YlfA and YlfB.
Fig 4.
YlfA and YlfB bind homo- and heterotypically in vitro.
A) Lysates from E. coli co-transformed with plasmids encoding GST and M45-tagged Ylfs were incubated with GS4B resin. Bound proteins were eluted with reduced glutathione and eluates were analyzed by anti-M45 epitope immunoblot. To assess the relative efficiency of binding and elution of GST and GST-Ylf fusion proteins, PVDF membranes were subsequently stained with Ponceau S. B) Whole cell lysates of stationary phase L. pneumophila were separated by SDS-PAGE and immobilized on PVDF. L. pneumophila strains include wild type (lane 1), in-frame deletion ΔylfA and ΔylfB mutants (lanes 2, 3), a double in-frame deletion ΔylfA ΔylfB mutant (lane 4), and the ΔylfA ΔylfB mutant harboring plasmids encoding M45-YlfA or M45-YlfB (lanes 5, 6.) Membranes were incubated with recombinant purified GST, GST-YlfA 128–425, or GST-YlfB 131–405 suspended at 20 μg/ml in 2% NFDM 0.1% Tween PBS overnight at 4°C. Bound GST or GST fusion protein was detected by anti-GST immunoblot.
Fig 5.
Purified YlfA and YlfB form high molecular weight complexes that are resistant to boiling.
6xHis-tagged YlfA 128–425 and YlfB 131–405 were applied to a Superdex 200 column. Peak fractions of each protein were combined with Laemmli buffer containing β-mercaptoethanol and a subset were heated for 10 min at 100°C. Protein complexes were detected by anti-YlfA and anti-YlfB immunoblot. Arrows indicate bands corresponding to monomeric, dimeric and trimeric species.
Fig 6.
YlfA and YlfB are required for efficient formation of the L. pneumophila-containing vacuole.
A) Bone marrow-derived macrophages derived from A/J mice were infected with wild type L. pneumophila, a ΔylfA ΔylfB mutant, and the ΔylfA ΔylfB mutant complemented by knock-in of both ylf ORFs onto the L. pneumophila chromosome. Duplicate samples were fixed at 2 and 10 h post-infection. The index of replicative vacuole formation was calculated by dividing the number of observed replicative vacuoles at 10 h by the total number of infected cells at 2 h. Due to variability in the overall efficiency of infection between individual experiments, the index for each strain is presented as a percent of wild type. Data represent the average of three independent experiments ± SD, n = 500 per time point, per experiment. ΔylfA ΔylfB mutant R.V. formation is significantly less efficient compared to wild type in an unpaired Student’s t test.*p<0.0001. B) Whole cell lysates were prepared from stationary phase broth cultures of wild type L. pneumophila, ΔylfA, ΔylfB, ΔylfA ΔylfB, and ΔylfA ΔylfB in which one or both ylf open reading frames have been reintroduced into the genome. YlfA and YlfB protein levels in these strains were detected by anti-YlfA and anti-YlfB immunoblot. Relative loading efficiency was monitored by anti-DotA immunoblot.
Fig 7.
The Ylfs are required for efficient recruitment of ER-derived membrane to LCVs.
PMA-differentiated U937 cells were infected with GFP-producing wild type, ΔdotA, ΔylfA ΔylfB, and ylf double knock-in strains of L. pneumophila. After 1 h of infection, L. pneumophila-containing vacuoles were recovered from the supernatant of infected cell lysates following low speed centrifugation. Vacuoles were stained with either anti-Calnexin or anti-LAMP1 antibodies to reveal interaction with host cell ER or endosomal compartments. Data represent the average of three independent experiments ± SD, n = 300 per condition, per experiment. ΔylfA ΔylfB mutant LCV Calnexin staining is significantly different compared to wild type in an unpaired Student’s t test.*p = 0.0001.
Table 1.
Strains, plasmids and primers.