The GARP complex is required for cellular sphingolipid homeostasis
- Harvard T.H. Chan School of Public Health, United States
- Harvard Medical School, United States
- University of Southern Denmark, Denmark
- Yale School of Medicine, United States
- Broad Institute, United States
- Howard Hughes Medical Institute, Harvard T.H. Chan School of Public Health, United States
Figures
Figure 1 with 1 supplement
A chemical biology screen reveals the retrograde endosome to Golgi trafficking machinery as a key regulator of sphingolipid homeostasis.
(A) A chemical genetic screen for interactions with myriocin. Calculated T-scores are plotted against colony size on control plates. Genes are color-coded according to their significance score. Red …
Figure 1—figure supplement 1
GO analysis of all suppressing mutants from the chemical genomic myriocin screen.
Gene ontology (GO) analysis of the hits obtained in our genome-wide chemical genetic screen is shown. Note, the GARP complex is strongly enriched among the suppressor mutants identified (p < 10−5), …
Figure 2 with 2 supplements
The disruption of the GARP complex leads to the accumulation of early sphingolipid synthesis intermediates.
(A, B, C) Blocking early steps of sphingolipid synthesis exacerbates GARP-associated growth defects. (A) GARP mutants are sensitive to IPC synthase inhibition. Wild-type, vps51Δ, vps52Δ, vps53Δ, and …
Figure 2—figure supplement 1
Model of Sphingolipid metabolism.
(A) Schematic of sphingolipid synthesis is shown. ER resident enzymes are indicated in blue. Golgi resident enzymes are indicated in yellow. Enzymes with multiple localizations are indicated in …
Figure 2—figure supplement 2
The rate of serine palimtoyl-transferase inhibition is similar in WT and vps53∆ cells.
Time-dependent response of DHS and PHS from wild-type (white lines) and vps53Δ cells (black lines) to myriocin treatment is plotted as fold change from time point 0.
Figure 3 with 1 supplement
GARP mutants show altered vacuolar morphology and function.
(A) Sphingolipid recycling remains blocked upon myriocin treatment and sphingolipids accumulate in vacuoles. Wild-type cells (top panels), wild-type cells treated with myriocin (1 μM, 12hr), vps53Δ …
Figure 3—figure supplement 1
Classification of vacuolar phenotypes used for quantification.
Vacuoles labeled with Vma1-mars (left panels), bright field images (middle panels), and merged images (right panels) are shown. Phenotypes were classified based on severity. Cells with 1–3 round …
Figure 4 with 1 supplement
The disruption of the GARP complex alters sterol distribution in yeast.
(A) Intracellular sterols accumulate in the GARP mutants vps53Δ and vps54Δ. Wild-type (left panels), vps53Δ cells (middle panels), and vps54Δ cell (right panels) treated with methanol (top panels) …
Figure 4—figure supplement 1
Components of the GARP complex genetically interact with ergosterol metabolism genes in yeast.
Tetrad analysis of vps52Δ mutants crossed with erg3Δ mutants is shown.
Figure 5 with 1 supplement
A PCCA2-causing GARP complex mutation is a partial loss of function allele.
(A) A mutation analogous to the VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans is partially resistant to sphingolipid biosynthesis inhibition induced by …
Figure 5—figure supplement 1
A GARP complex mutation analogous to the VPS53 allele causing PCCA2 vps53 Q624R does not impact protein stability.
vps53Δ cells harboring a plasmid encoding vps53 Q624R or wild-type Vps53 were labeled with either light or heavy L-lysine. Protein intensities are plotted against light/heavy SILAC ratios. …
Figure 6 with 2 supplements
The depletion of sphingolipid levels reduced lysosome clustering and sterol accumulation due to GARP complex deficiency in HeLa cells.
(A, B) Myriocin treatment reduced build-up of free cholesterol due to GARP complex deficiency. (A) Filipin staining of unesterified cholesterol in control (top panels) or Vps53 knock-down (lower …
Figure 6—figure supplement 1
Myriocin treatment reduced accumulations of early sphingolipid intermediates due to GARP complex deficiency.
Lipidomic analysis of mock-treated HeLa control cells (white bars), myriocin-treated control cells (light gray bars), mock-treated VPS53 KD cells (dark gray bars), and myriocin-treated VPS53 KD …
Figure 6—figure supplement 2
LAMP1 expression in VPS53 knock-down HeLa cells is not altered.
HeLa control cells and HeLa VPS53 KD cells were analyzed by Western blotting against VPS53, LAMP1, or tubulin as control.
Additional files
-
Supplementary file 1
List of all yeast strains used in this study.
- https://doi.org/10.7554/eLife.08712.018
-
Supplementary file 2
List of all plasmids used in this study.
- https://doi.org/10.7554/eLife.08712.019
-
Supplementary file 3
List of all hits identified in the chemical genomic screen.
- https://doi.org/10.7554/eLife.08712.020
-
Supplementary file 4
Phenotypes of different trafficking complexes identified in the chemical genetic screen.
- https://doi.org/10.7554/eLife.08712.021
