Limits...
Phosphoinositide kinase signaling controls ER-PM cross-talk.

Omnus DJ, Manford AG, Bader JM, Emr SD, Stefan CJ - Mol. Biol. Cell (2016)

Bottom Line: However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress.We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress.Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

ER-PM cross-talk controls ceramide synthase activity in the ER. (A) Synthesis of ceramides, but not LCBs, is compromised in the Δtether cells. Wild-type, Δtether, sac1Δ, and orm1Δ orm2Δ cells were labeled with [3H]serine for 60 min at 26°C. Sphingolipids were extracted and analyzed by TLC. LCBs, phosphorylated-LCBs (LCB-P), ceramides (Cer), and other sphingolipid species are indicated. The hatched ovals point out elevated LCBs and phosphorylated LCBs in the Δtether mutant cells. In addition, note that ceramides are reduced in the Δtether mutant cells as compared with wild-type cells (Figure 2). The sac1Δ and orm1Δ orm2Δ cells serve as controls for LCBs and phosphorylated LCBs. See Supplemental Figure S6, A and B. (B) Schematic cartoon depicting topology of Lac1 and Lag1 in the ER. Both proteins possess Ypk1/2 consensus sites in their N-terminal cytoplasmic tails. (C) Double-mutant lac1Δ lag1ts cells expressing either wild-type Lag1 or the phosphomimetic form Lag1S23,24E from plasmids were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. (D) Analysis of Lag1-GFP phosphorylation upon heat stress conditions. Wild-type, Δtether, and ypk1ts ypk2Δ cells expressing Lag1-GFP were incubated at 38°C for 2 h. Protein extracts were prepared, separated by Phos-tag gel electrophoresis, and analyzed by immunoblotting using an antibody that specifically recognizes GFP. Wild-type cells carrying empty vector were included as a control for the GFP antisera. SDS–PAGE and immunoblotting were used to determine expressions levels of PGK as a protein loading control. (E) Quantitation of phosphorylated Lag1-GFP level in wild-type and Δtether cells as a percentage of total Lag1-GFP protein levels. The data represent means ± SDs from two independent experiments analyzed in duplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4814223&req=5

Figure 6: ER-PM cross-talk controls ceramide synthase activity in the ER. (A) Synthesis of ceramides, but not LCBs, is compromised in the Δtether cells. Wild-type, Δtether, sac1Δ, and orm1Δ orm2Δ cells were labeled with [3H]serine for 60 min at 26°C. Sphingolipids were extracted and analyzed by TLC. LCBs, phosphorylated-LCBs (LCB-P), ceramides (Cer), and other sphingolipid species are indicated. The hatched ovals point out elevated LCBs and phosphorylated LCBs in the Δtether mutant cells. In addition, note that ceramides are reduced in the Δtether mutant cells as compared with wild-type cells (Figure 2). The sac1Δ and orm1Δ orm2Δ cells serve as controls for LCBs and phosphorylated LCBs. See Supplemental Figure S6, A and B. (B) Schematic cartoon depicting topology of Lac1 and Lag1 in the ER. Both proteins possess Ypk1/2 consensus sites in their N-terminal cytoplasmic tails. (C) Double-mutant lac1Δ lag1ts cells expressing either wild-type Lag1 or the phosphomimetic form Lag1S23,24E from plasmids were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. (D) Analysis of Lag1-GFP phosphorylation upon heat stress conditions. Wild-type, Δtether, and ypk1ts ypk2Δ cells expressing Lag1-GFP were incubated at 38°C for 2 h. Protein extracts were prepared, separated by Phos-tag gel electrophoresis, and analyzed by immunoblotting using an antibody that specifically recognizes GFP. Wild-type cells carrying empty vector were included as a control for the GFP antisera. SDS–PAGE and immunoblotting were used to determine expressions levels of PGK as a protein loading control. (E) Quantitation of phosphorylated Lag1-GFP level in wild-type and Δtether cells as a percentage of total Lag1-GFP protein levels. The data represent means ± SDs from two independent experiments analyzed in duplicate.

Mentions: LCBs have also been suggested to regulate Pkh1/2 function in vivo (Liu et al., 2005), and LCBs are elevated in cells lacking the Sac1 PI4P phosphatase activity (see later discussion of Figure 6A; Brice et al., 2009; Breslow et al., 2010). However, GFP-Pkh1 remained localized to punctate structures in sac1 mutant cells treated with myriocin, which blocks LCB synthesis (Supplemental Figure S4D; 3–10 puncta/cell), indicating that increased PI4P levels may be sufficient for Pkh1 membrane recruitment. Consistent with this, the intensity of GFP-Pkh1 assemblies decreased in stt4ts sac1Δ double mutant cells as compared with sac1Δ single-mutant cells (Supplemental Figure S4E). Moreover, GFP-Pkh1 puncta were not significantly increased at 26 or 42°C in orm1 orm2 mutant cells, which have elevated LCB levels (Supplemental Figure S4F and Supplemental Table S3; see later discussion of Figure 6A; Brice et al., 2009; Breslow et al., 2010).


Phosphoinositide kinase signaling controls ER-PM cross-talk.

Omnus DJ, Manford AG, Bader JM, Emr SD, Stefan CJ - Mol. Biol. Cell (2016)

ER-PM cross-talk controls ceramide synthase activity in the ER. (A) Synthesis of ceramides, but not LCBs, is compromised in the Δtether cells. Wild-type, Δtether, sac1Δ, and orm1Δ orm2Δ cells were labeled with [3H]serine for 60 min at 26°C. Sphingolipids were extracted and analyzed by TLC. LCBs, phosphorylated-LCBs (LCB-P), ceramides (Cer), and other sphingolipid species are indicated. The hatched ovals point out elevated LCBs and phosphorylated LCBs in the Δtether mutant cells. In addition, note that ceramides are reduced in the Δtether mutant cells as compared with wild-type cells (Figure 2). The sac1Δ and orm1Δ orm2Δ cells serve as controls for LCBs and phosphorylated LCBs. See Supplemental Figure S6, A and B. (B) Schematic cartoon depicting topology of Lac1 and Lag1 in the ER. Both proteins possess Ypk1/2 consensus sites in their N-terminal cytoplasmic tails. (C) Double-mutant lac1Δ lag1ts cells expressing either wild-type Lag1 or the phosphomimetic form Lag1S23,24E from plasmids were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. (D) Analysis of Lag1-GFP phosphorylation upon heat stress conditions. Wild-type, Δtether, and ypk1ts ypk2Δ cells expressing Lag1-GFP were incubated at 38°C for 2 h. Protein extracts were prepared, separated by Phos-tag gel electrophoresis, and analyzed by immunoblotting using an antibody that specifically recognizes GFP. Wild-type cells carrying empty vector were included as a control for the GFP antisera. SDS–PAGE and immunoblotting were used to determine expressions levels of PGK as a protein loading control. (E) Quantitation of phosphorylated Lag1-GFP level in wild-type and Δtether cells as a percentage of total Lag1-GFP protein levels. The data represent means ± SDs from two independent experiments analyzed in duplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4814223&req=5

Figure 6: ER-PM cross-talk controls ceramide synthase activity in the ER. (A) Synthesis of ceramides, but not LCBs, is compromised in the Δtether cells. Wild-type, Δtether, sac1Δ, and orm1Δ orm2Δ cells were labeled with [3H]serine for 60 min at 26°C. Sphingolipids were extracted and analyzed by TLC. LCBs, phosphorylated-LCBs (LCB-P), ceramides (Cer), and other sphingolipid species are indicated. The hatched ovals point out elevated LCBs and phosphorylated LCBs in the Δtether mutant cells. In addition, note that ceramides are reduced in the Δtether mutant cells as compared with wild-type cells (Figure 2). The sac1Δ and orm1Δ orm2Δ cells serve as controls for LCBs and phosphorylated LCBs. See Supplemental Figure S6, A and B. (B) Schematic cartoon depicting topology of Lac1 and Lag1 in the ER. Both proteins possess Ypk1/2 consensus sites in their N-terminal cytoplasmic tails. (C) Double-mutant lac1Δ lag1ts cells expressing either wild-type Lag1 or the phosphomimetic form Lag1S23,24E from plasmids were preincubated at the appropriate temperature for 10 min and labeled with [3H]serine for 60 min. Sphingolipids were extracted and analyzed by TLC. (D) Analysis of Lag1-GFP phosphorylation upon heat stress conditions. Wild-type, Δtether, and ypk1ts ypk2Δ cells expressing Lag1-GFP were incubated at 38°C for 2 h. Protein extracts were prepared, separated by Phos-tag gel electrophoresis, and analyzed by immunoblotting using an antibody that specifically recognizes GFP. Wild-type cells carrying empty vector were included as a control for the GFP antisera. SDS–PAGE and immunoblotting were used to determine expressions levels of PGK as a protein loading control. (E) Quantitation of phosphorylated Lag1-GFP level in wild-type and Δtether cells as a percentage of total Lag1-GFP protein levels. The data represent means ± SDs from two independent experiments analyzed in duplicate.
Mentions: LCBs have also been suggested to regulate Pkh1/2 function in vivo (Liu et al., 2005), and LCBs are elevated in cells lacking the Sac1 PI4P phosphatase activity (see later discussion of Figure 6A; Brice et al., 2009; Breslow et al., 2010). However, GFP-Pkh1 remained localized to punctate structures in sac1 mutant cells treated with myriocin, which blocks LCB synthesis (Supplemental Figure S4D; 3–10 puncta/cell), indicating that increased PI4P levels may be sufficient for Pkh1 membrane recruitment. Consistent with this, the intensity of GFP-Pkh1 assemblies decreased in stt4ts sac1Δ double mutant cells as compared with sac1Δ single-mutant cells (Supplemental Figure S4E). Moreover, GFP-Pkh1 puncta were not significantly increased at 26 or 42°C in orm1 orm2 mutant cells, which have elevated LCB levels (Supplemental Figure S4F and Supplemental Table S3; see later discussion of Figure 6A; Brice et al., 2009; Breslow et al., 2010).

Bottom Line: However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress.We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca(2+)-regulated lipid biogenesis, upon plasma membrane (PM) stress.Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory for Molecular Cell Biology, University College London, London WC1E 6BT, United Kingdom.

No MeSH data available.


Related in: MedlinePlus