Lysophosphatidylcholine Acyltransferase 1 (LPCAT1) Specifically Interacts with Phospholipid Transfer Protein StarD10 to Facilitate Surfactant Phospholipid Trafficking in Alveolar Type II Cells

Abstract

Pulmonary surfactant, a mixture of proteins and phospholipids, plays an important role in facilitating gas exchange by maintaining alveolar stability. Saturated phosphatidylcholine (SatPC), the major component of surfactant, is synthesized both de novo and by the remodeling of unsaturated phosphatidylcholine (PC) by lyso-PC acyltransferase 1 (LPCAT1). After synthesis in the endoplasmic reticulum, SatPC is routed to lamellar bodies (LBs) for storage prior to secretion. The mechanism by which SatPC is transported to LB is not understood. The specificity of LPCAT1 for lyso-PC as an acyl acceptor suggests that formation of SatPC via LPCAT1 reacylation is a final step in SatPC synthesis prior to transport. We hypothesized that LPCAT1 forms a transient complex with SatPC and specific phospholipid transport protein(s) to initiate trafficking of SatPC from the endoplasmic reticulum to the LB. Herein we have assessed the ability of different StarD proteins to interact with LPCAT1. We found that LPCAT1 interacts with StarD10, that this interaction is direct, and that amino acids 79-271 of LPCAT1 and the steroidogenic acute regulatory protein-related lipid transfer (START) domain of START domain-containing protein 10 (StarD10) are sufficient for this interaction. The role of StarD10 in trafficking of phospholipid to LB was confirmed by the observation that knockdown of StarD10 significantly reduced transport of phospholipid to LB. LPCAT1 also interacted with one isoform of StarD7 but showed no interaction with StarD2/PC transfer protein.

Keywords: lipid trafficking; lipid-binding protein; phospholipid; protein expression; protein-protein interaction.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Ontogeny of StarD10, StarD2/PCTP, StarD7, and LPCAT1 in the developing mouse lung.A–D, qPCR analysis of StarD10 (A), StarD2/PCTP (B), StarD7 (C), and LPCAT1 (D) expression in whole mouse lungs from stages E11.5 to adult. The mRNA level of each gene was normalized to β-actin at each time point. Note the differences in scale on the graphs for relative expression of the target genes. Data were derived from three independent litters at each time point. E, comparison of StarD2/PCTP, StarD7, StarD10, and LPCAT1 expression in freshly isolated adult mouse alveolar type II cells. StarD10 abundance was 31 times that of StarD2/PCTP and 8 times that of StarD7. Note that the level of LPCAT1 mRNA was most abundant among these four genes in the type II cells. Error bars represent S.E.

FIGURE 2.

LPCAT1 interacts directly with StarD10.A, schematic of HA- or FLAG-tagged constructs for full-length mouse LPCAT1 and StarD10. Co-IP of LPCAT-HA plus StarD10-FLAG (B) and LPCAT1-FLAG plus StarD10-HA (C) is shown. Total lysates of HEK293 cells were singly transfected with empty vector, LPCAT1, or StarD10 or co-transfected with both LPCAT1 and StarD10; immunoprecipitated with resins conjugated with anti-FLAG antibody; and immunoblotted with anti-HA antibody. 5% of cell lysates was used as input; expression of HA-tagged prey and FLAG-tagged bait protein was demonstrated by immunoblotting with anti-HA and anti-FLAG antibodies, respectively. Note that HA-tagged LPCAT1 or StarD10 was pulled down by FLAG-tagged StarD10 or LPCAT1, respectively, demonstrating that co-IP with LPCAT1 and StarD10 worked in both directions. Molecular sizes are indicated in kDa. Results are representative of four independent experiments. D, schematic of GST- or His-tagged mouse LPCAT1 and StarD10 constructs used for the production of recombinant proteins. GST pulldown assays for GST-LPCAT1 plus StarD10-His (E) and LPCAT1-His plus GST-StarD10 (F) are shown. GST or His column-purified recombinant LPCAT1 and/or StarD10 was incubated with GST-resin followed by immunoblotting against LPCAT1 and StarD10. Note that StarD10-His was pulled down by GST-LPCAT1 and that LPCAT-His was pulled down by GST-StarD10, demonstrating that the interaction of LPCAT1 with StarD10 was unaffected by the position of the GST and His tags. Results are representative of four independent experiments.

FIGURE 3.

LPCAT1 interacts with StarD10 in vivo.A, cryostat sections of frozen fixed adult lungs were subjected to in situ PLA using antibodies against LPCAT1 and StarD10. Confocal images of the PLA signal (red dots; arrows) shows that LPCAT1 and StarD10 interact in wild-type mouse lung (top row). No signal is apparent in sections of lungs from Lpcat1−/− mice (middle row) or in sections in which normal rabbit IgG was substituted for anti-LPCAT1 (negative control; bottom row). The images are representative of lung sections from two different mice for each group in five independent experiments. Scale bar, 10 μm. B, quantification of PLA signals. The values represent the mean ± S.E. (error bars) of five independent experiments in each group. p < 0.0001 versus Lpcat1−/− lungs and the negative control. NS, not significant (p ≥ 0.05).

FIGURE 4.

Interactions between LPCAT1 mutants and full-length StarD10.A, schematic of HA-tagged LPCAT1 truncated mutants and full-length StarD10-FLAG constructs. B, co-IP of LPCAT1(49–534)-HA plus StarD10-FLAG resulted in pulldown of LPCAT1(49–534) by StarD10. C, co-transfection of LPCAT1-HA and LPCAT1(49–534)-HA with StarD10-FLAG resulted in pulldown of both full-length and truncated LPCAT1 by StarD10. Note that the expression of full-length LPCAT1 was slightly higher than that of truncated LPCAT1(49–534). D, co-IP of LPCAT1(79–534)-HA plus StarD10-FLAG resulted in pulldown of LPCAT1(79–534) by StarD10. NS, nonspecific band (∼35 kDa). E, co-transfection of LPCAT(79–534)-HA and LPCAT1(272–534)-HA with StarD10-FLAG resulted in pulldown of only LPCAT1(79–534), suggesting that the region of LPCAT1 that interacts with StarD10 is found between amino acids 79 and 271. Note that expression of LPCAT1(272–534) is higher than that of LPCAT(79–534). Results are representative of three independent experiments.

FIGURE 5.

Amino acids 79–271 are required for interaction of LPCAT1 with StarD10.A, schematic of HA-tagged LPCAT1 mutants and full-length StarD10-FLAG constructs. B, co-IP of LPCAT1(1–271)-HA plus StarD10-FLAG showing that this fragment interacts with StarD10. C, co-IP analysis shows that mutant LPCAT1(79–271)-HA, a further truncation of LPCAT1, also interacts with StarD10-FLAG. NS, nonspecific band. Results are representative of three independent experiments.

FIGURE 6.

Topology of LPCAT1.A, schematic of N-terminal FLAG-tagged or C-terminal HA-tagged full-length LPCAT1 constructs. B, proteinase K protection assay. Microsomes isolated from HEK293 cells transiently expressing FLAG- or HA-tagged LPCAT1 were incubated with buffer only, proteinase K, or proteinase K plus Triton X-100 (Prot. K + Triton) followed by immunoblotting with anti-HA or anti-FLAG antibody. The lack of detection of the N terminus of LPCAT1 with anti-FLAG antibody and the C terminus with anti-HA antibody after proteinase K digestion indicates that both termini were exposed on the exterior of the microsome. The integrity of microsomes was demonstrated by immunoblotting against the luminal ER marker BiP. C, model depicting the monotopic orientation of LPCAT1. LPCAT1 is attached to the ER membrane through its TMD (red) with both the N and C termini facing the cytosol.

FIGURE 7.

The START domain of StarD10 is required for interaction with LPCAT1.A, schematic of HA- or FLAG-tagged full-length LPCAT1 and mutant StarD10 constructs. B and C, co-IP of LPCAT1 and StarD10 mutants. Co-IP of LPCAT1-FLAG plus StarD10(1–227)-HA resulted in pulldown of StarD10(1–227) by LPCAT1 (B), whereas co-IP of LPCAT1-HA plus StarD10(19–227)-FLAG resulted in pulldown of LPCAT1 by StarD10(19–227) (C), demonstrating that the START domain of StarD10 is sufficient for its interaction with LPCAT1. NS, nonspecific band. Results are representative of three independent experiments.

FIGURE 8.

Knockdown of StarD10 results in decreased PC transport to LB.A, qPCR analysis of StarD10 mRNA in mouse type II cells treated with siRNAs targeting StarD10. Freshly isolated mouse type II cells were cultured and transfected with the viral vector alone, scrambled siRNA, and various StarD10 Stealth RNAi siRNAs. The level of endogenous StarD10 mRNA was quantitated by qPCR and normalized to β-actin. Note that StarD10 siRNA 3 was more effective in knocking down StarD10 than siRNA 1, siRNA 2, or a combination of all three. The data represent the mean ± S.E. (error bars) of three independent experiments. *, p < 0.0001 versus scrambled siRNA. B, immunoblot analysis of StarD10 protein levels in whole cell lysates treated with StarD10 siRNA. StarD10 protein was significantly reduced in cells treated with StarD10 siRNA compared with scrambled siRNA. NS, nonspecific band. Results are representative of three independent experiments. C, 72 h after siRNA treatment, type II cells were labeled for 16 h with NBD-palmitoyl-CoA, then LBs were isolated, and the fluorescent content was determined. Incorporation of fluorescent palmitoyl-CoA-labeled phospholipid into LB was normalized to LB protein content. The amount of fluorescent phospholipid that was transported to LB was significantly reduced in cells transfected with StarD10 siRNA compared with scrambled siRNA. The data represent the mean ± S.E. (error bars) of six independent experiments. *, p = 0.0013 versus scrambled siRNA. D, mouse type II cells treated with scrambled or StarD10 siRNA were labeled with [3H]PA, and incorporation into SatPC was determined. [3H]PA incorporation into SatPC was not significantly different in cells treated with scrambled or StarD10 siRNA, indicating that SatPC synthesis itself was unaffected. No change in the amount of SatPC released into the medium was detected as well. The data represent the mean ± S.E. (error bars) of three independent experiments.

FIGURE 9.

LPCAT1 interacts with StarD7-I through its N terminus but does not interact with StarD2/PCTP.A, schematic of FLAG- or HA-tagged LPCAT1, StarD7, and StarD2/PCTP constructs. B, co-IP of LPCAT1-FLAG and StarD7-I-HA. Co-IP using LPCAT1-FLAG and StarD7-I-HA resulted in pulldown of StarD7-I by LPCAT1. The alternative splicing variant StarD7-II was detected but did not co-precipitate with LPCAT1. C, co-IP of LPCAT1-HA and StarD7-I(1–78)-FLAG. Co-IP with LPCAT1-HA and StarD7-I(1–78)-FLAG resulted in pulldown of LPCAT1 by StarD7-I(1–78), demonstrating that the N-terminal 78 amino acids of StarD7-I are sufficient for interaction with LPCAT1. NS, nonspecific band. D, StarD2/PCTP does not interact with LPCAT1. These data represent the results of five independent experiments.