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. 2013 Mar;54(3):831-842.
doi: 10.1194/jlr.M034678. Epub 2013 Jan 16.

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids

Keisuke Obara  1 , Ryo Kojima  1 , Akio Kihara  1
Affiliations

Effects on vesicular transport pathways at the late endosome in cells with limited very long-chain fatty acids

Keisuke Obara et al. J Lipid Res. 2013 Mar.

Abstract

Very long-chain fatty acids (VLCFAs), fatty acids with chain-length greater than 20 carbons, possess a wide range of biological functions. However, their roles at the molecular level remain largely unknown. In the present study, we screened for multicopy suppressors that rescued temperature-sensitive growth of VLCFA-limited yeast cells, and we identified the VPS21 gene, encoding a Rab GTPase, as such a suppressor. When the vps21Δ mutation was introduced into a deletion mutant of the SUR4 gene, which encodes a VLCFA elongase, a synthetic growth defect was observed. Endosome-mediated vesicular trafficking pathways, including endocytosis and the carboxypeptidase Y (CPY) pathway, were severely impaired in sur4Δ vps21Δ double mutants, while the AP-3 pathway that bypasses the endosome was unaffected. In addition, the sur4Δ mutant also exhibited a synthetic growth defect when combined with the deletion of VPS3, which encodes a subunit of the class C core vacuole/endosome tethering (CORVET) complex that tethers transport vesicles to the late endosome/multivesicular body (MVB). These results suggest that, of all the intracellular trafficking pathways, requirement of VLCFAs is especially high in the endosomal pathways.

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Figures

Fig. 1.
Fig. 1.
Cells’ requirement of VLCFA is significant at elevated temperature. (A) BY4741 (wild-type), 5281 (sur4Δ), 5763 (fen1Δ), YOK2230 (PtetO7-PHS1), MRY9 (PtetO7-PHS1 sur4Δ), and MRY7 (PtetO7-PHS1 fen1Δ) cells were grown to stationary phase, serially diluted at 1:10, spotted on YPD plates, grown for the indicated times at 30°C or 39°C, and photographed against a dark background. (B) YOK2230 (PtetO7-PHS1) and MRY9 (PtetO7-PHS1 sur4Δ) cells were grown to log phase at 30°C, and total membrane fractions were prepared. Samples (20 μg) were incubated with 20 μM palmitoyl-CoA, 73 μM malonyl-CoA, and 27.3 μM [14C]malonyl-CoA (0.075 μCi) for 1 h at 37°C. Lipids were saponified, acidified, extracted, converted to methyl ester forms, separated by reverse-phase TLC, and detected by autoradiography.
Fig. 2.
Fig. 2.
Deletion of the SUR4 and VPS21 genes results in synthetic growth defect. (A) BY4741 (wild-type), 1865 (vps21Δ), 5281 (sur4Δ), MRY91 (sur4Δ vps21Δ), 5763 (fen1Δ), and MRY90 (fen1Δ vps21Δ) cells were grown to stationary phase, serially diluted at 1:10, spotted on YPD plates, grown for indicated time at 30°C, 36°C, or 39°C, and photographed against a dark background. (B) Total membrane fractions (20 μg) prepared from BY4741 (wild-type), 1865 (vps21Δ), 5281 (sur4Δ), MRY91 (sur4Δ vps21Δ), 5763 (fen1Δ), and MRY90 (fen1Δ vps21Δ) cells grown at 30°C were incubated with 20 μM palmitoyl-CoA, 73 μM malonyl-CoA, and 27.3 μM [14C]malonyl-CoA (0.075 μCi) for 1 h at 37°C. Lipids were saponified, acidified, extracted, converted to methyl ester forms, separated by reverse-phase TLC, and detected by autoradiography.
Fig. 3.
Fig. 3.
The CPY pathway is affected in sur4Δ vps21Δ and fen1Δ vps21Δ mutants. BY4741 (wild-type), MRY90 (fen1Δ vps21Δ), MRY91 (sur4Δ vps21Δ), 1865 (vps21Δ), 5763 (fen1Δ), and 5281 (sur4Δ) cells were grown at 30°C to log phase. (A) Intracellular total lysates (I) and extracellular proteins recovered from the medium (E) were separated by SDS-PAGE, followed by immunoblotting with an anti-CPY antibody. (B) Cells were pulse-labeled with 100 μCi [35S]methionine/cysteine for 15 min and chased for 20 min. Total lysates were prepared, and CPY was immunoprecipitated with an anti-CPY antibody. Precipitates were subjected to SDS-PAGE and detected using a bioimaging analyzer BAS-2500. (C) Total lysates were prepared, separated by SDS-PAGE, and detected by immunoblotting with an anti-Pep4, anti-Pho8, or to demonstrate uniform protein loading, anti-Pgk1 antibody. m, mature form; pro, proform; s, soluble mature form.
Fig. 4.
Fig. 4.
Transport of Cps1 to the vacuole is impaired in sur4Δ vps21Δ and fen1Δ vps21Δ mutants. (A) BY4741 (wild-type), 5763 (fen1Δ), 5281 (sur4Δ), 1865 (vps21Δ), MRY90 (fen1Δ vps21Δ), and MRY91 (sur4Δ vps21Δ) cells, each harboring pOK489 (mRFP-Cps1), were grown at 30°C to log phase and subjected to fluorescence microscopy. Left panels, DIC images; right panels, mRFP fluorescence. Bar, 5 μm. (B) Total lysates were prepared from cells indicated in (A), separated by SDS-PAGE, and detected by immunoblotting with anti-DsRed antibody or, to demonstrate uniform protein loading, anti-Pgk1 antibody. Expression levels of mRFP-Cps1 were significantly low in the fen1Δ and sur4Δ mutants, compared with the other strains used, for an unknown reason. Thus, the amounts of lysates subjected to SDS-PAGE were increased for the fen1Δ and sur4Δ mutants (3- and 4-fold for the fen1Δ and sur4Δ cells, respectively) to make the total amount of mRFP-Cps1 plus mRFP comparable to the other strains.
Fig. 5.
Fig. 5.
Endocytosis of Ste2 is impaired in sur4Δ vps21Δ and fen1Δ vps21Δ cells. (A) BY4741 (wild-type), 5763 (fen1Δ), 5281 (sur4Δ), 1865 (vps21Δ), MRY90 (fen1Δ vps21Δ), and MRY91 (sur4Δ vps21Δ) cells, each harboring pOK529 (Ste2-mRFP), were grown at 30°C to log phase and subjected to fluorescence microscopy. Left panels, DIC images; right panels, mRFP fluorescence. Bar, 5 μm. (B) Total lysates were prepared from cells indicated in (A) and separated by SDS-PAGE, followed by immunoblotting with anti-DsRed antibody or, to demonstrate uniform protein loading, anti-Pgk1 antibody. The lower panel represents the same immunoblot as the middle panel but a longer exposure to X-ray film performed to enhance the image.
Fig. 6.
Fig. 6.
Sphingolipids containing VLCFAs cooperate with Vps21 in vesicular transport pathways that traffic to the vacuole. (A) BY4741 (wild-type), 1865 (vps21Δ), 4007 (ipt1Δ), and MRY134 (ipt1Δ vps21Δ) cells were grown to stationary phase, serially diluted at 1:10, spotted on YPD plates, grown for 48 h at 30°C or 36°C, and photographed against a dark background. (B) BY4741 (wild-type), 1865 (vps21Δ), MRY160 (csg1Δ csh1Δ vps21Δ), MRY129 (csg1Δ csh1Δ), MRY157 (csg1Δ vps21Δ), 2771 (csg1Δ), MRY159 (csh1Δ vps21Δ), and 3300 (csh1Δ) cells were grown to stationary phase, serially diluted at 1:10, spotted on YPD plates, grown for 48 h at 30°C or 36°C, and photographed against a dark background. (C) BY4741 (wild-type), 1865 (vps21Δ), MRY129 (csg1Δ csh1Δ), and MRY160 (csg1Δ csh1Δ vps21Δ) cells, each harboring pOK489 (mRFP-Cps1) or pOK529 (Ste2-mRFP), were grown at 30°C to log phase, then subjected to fluorescence microscopy. Left panels, DIC images; right panels, mRFP fluorescence. Bars, 5 μm. (D) BY4741 (wild-type), MRY160 (csg1Δ csh1Δ vps21Δ), 1865 (vps21Δ), and MRY129 (csg1Δ csh1Δ) cells, alone or transfected with pOK489 (mRFP-Cps1), were grown at 30°C to log phase. Total lysates were extracted and separated by SDS-PAGE, and proteins were detected by immunoblotting with an anti-DsRed, anti-CPY, anti-Pep4, anti-Pho8, or to demonstrate uniform protein loading, anti-Pgk1 antibody. m, mature form; pro, proform.
Fig. 7.
Fig. 7.
The SUR4 gene interacts at the genetic level with genes encoding CORVET subunits. (A) BY4741 (wild-type), 5281 (sur4Δ), 4329 (vps3Δ), MRY121 (sur4Δ vps3Δ), 405 (vps8Δ), MRY135 (sur4Δ vps8Δ), 3774 (vps39Δ), MRY136 (sur4Δ vps39Δ), 4015 (vps41Δ), and MRY137 (sur4Δ vps41Δ) cells were grown to stationary phase, serially diluted at 1:10, spotted on YPD plates, grown for 48 h at 30°C or 36°C, and photographed against a dark background. (B) BY4741 (wild-type), 5281 (sur4Δ), MRY235 (vps8Δ vps19Δ), MRY242 (sur4Δ vps8Δ vps19Δ), MRY106 (vps19Δ), and MRY124 (sur4Δ vps19Δ) cells were grown to stationary phase, serially diluted at 1:10, spotted on YPD plates, grown for 48 h at 30°C or 36°C, and photographed against a dark background. (C) Schematic representation of the CORVET and HOPS complexes and interactions between their encoding genes and the SUR4 gene. Deletion of VPS21 or VPS3 causes severe synthetic growth defect when combined with the SUR4 deletion; these genes are illustrated as filled gray circles with thick lines. Simultaneous disruption of genes encoding the Vps21 effectors VPS8 and VPS19 also results in severe synthetic growth defect when combined with the SUR4 deletion; these genes are illustrated as white circles enclosed by thick lines. Two-headed arrows indicate protein-protein interactions between Rab GTPases and proteins involved in vesicle tethering.
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