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Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function.

Schmidt JA, Brown WJ - J. Cell Biol. (2009)

Bottom Line: Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro.Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA).These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid-specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

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LPAAT3 overexpression slows BFA and CI-976–stimulated Golgi retrograde trafficking to the ER. (a) HeLa cells and cells transfected with LPAAT3 WT or LPAAT3 MT were treated with 10 µg/ml BFA and analyzed by immunofluorescence with GPP130 as a Golgi marker. Control cells, LPAAT3 MT cells, and LPAAT6 cells formed Golgi membrane tubules from 2 to 5 min after addition of BFA and were completely relocalized to ER membranes within 10 min, whereas LPAAT3 WT cells were slower. Bar = 20 µm. (b) The t1/2 of Golgi loss due to BFA tubulation was 5.5, 5.0, and 4.0 min for HeLa cells, LPAAT3 MT cells, and LPAAT6 cells, respectively, and 11 min for LPAAT3 WT cells. (c) To measure LPAAT3 knockdown cells treated with BFA, the experiment above was repeated at 24°C where the t1/2 values for cells was 18 min for control cells, 25 min for cells overexpressing LPAAT3 WT, and 11.2 min for LPAAT3 knockdown cells. Data are representative of three independent experiments. (d) Control cells, LPAAT3 WT cells, LPAAT3 MT cells, and LPAAT3 siRNA knockdown cells were treated with 50 µM of the LPAT inhibitor CI-976 for 30 min. Cells expressing LPAAT3 WT were slower at forming Golgi membrane tubules, whereas knockdown cells were considerably faster. Bar = 20 µm. (e) Quantification of the CI-976 results. All error bars are SD for three independent experiments (n = 300).
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fig3: LPAAT3 overexpression slows BFA and CI-976–stimulated Golgi retrograde trafficking to the ER. (a) HeLa cells and cells transfected with LPAAT3 WT or LPAAT3 MT were treated with 10 µg/ml BFA and analyzed by immunofluorescence with GPP130 as a Golgi marker. Control cells, LPAAT3 MT cells, and LPAAT6 cells formed Golgi membrane tubules from 2 to 5 min after addition of BFA and were completely relocalized to ER membranes within 10 min, whereas LPAAT3 WT cells were slower. Bar = 20 µm. (b) The t1/2 of Golgi loss due to BFA tubulation was 5.5, 5.0, and 4.0 min for HeLa cells, LPAAT3 MT cells, and LPAAT6 cells, respectively, and 11 min for LPAAT3 WT cells. (c) To measure LPAAT3 knockdown cells treated with BFA, the experiment above was repeated at 24°C where the t1/2 values for cells was 18 min for control cells, 25 min for cells overexpressing LPAAT3 WT, and 11.2 min for LPAAT3 knockdown cells. Data are representative of three independent experiments. (d) Control cells, LPAAT3 WT cells, LPAAT3 MT cells, and LPAAT3 siRNA knockdown cells were treated with 50 µM of the LPAT inhibitor CI-976 for 30 min. Cells expressing LPAAT3 WT were slower at forming Golgi membrane tubules, whereas knockdown cells were considerably faster. Bar = 20 µm. (e) Quantification of the CI-976 results. All error bars are SD for three independent experiments (n = 300).

Mentions: Pharmacological studies have suggested that Golgi membrane tubules are positively influenced by PLA2 activity and negatively influenced by LPAT activity (Brown et al., 2003). Cells were all treated with the fungal metabolite brefeldin A (BFA), which generates numerous membrane tubules that redistribute Golgi membranes to the ER (Lippincott-Schwartz et al., 1989). Cells overexpressing LPAAT3 form Golgi tubules much slower than control and LPAAT3 MT cells (Fig. 3, a and b). Moreover, the Golgi tubules that did form in LPAAT3 WT overexpressing cells were far fewer in number and shorter in length than untransfected cells, as shown by Sholl analyses (Fig. S3, a and b; Sholl, 1953). The inhibition of BFA-stimulated tubule formation by LPAAT3 overexpression was not due to ineffective COPI disruption (Fig. S3 c). In contrast to overexpression, LPAAT3 knockdown cells exhibited a very rapid loss of the Golgi in BFA, so the experiment was repeated at 24°C to obtain quantitative data. The results showed that the half-time for loss of the Golgi in siRNA-treated cells was 11 min compared with 25 min for overexpressing cells (17 min in control cells) (Fig. 3 c).


Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function.

Schmidt JA, Brown WJ - J. Cell Biol. (2009)

LPAAT3 overexpression slows BFA and CI-976–stimulated Golgi retrograde trafficking to the ER. (a) HeLa cells and cells transfected with LPAAT3 WT or LPAAT3 MT were treated with 10 µg/ml BFA and analyzed by immunofluorescence with GPP130 as a Golgi marker. Control cells, LPAAT3 MT cells, and LPAAT6 cells formed Golgi membrane tubules from 2 to 5 min after addition of BFA and were completely relocalized to ER membranes within 10 min, whereas LPAAT3 WT cells were slower. Bar = 20 µm. (b) The t1/2 of Golgi loss due to BFA tubulation was 5.5, 5.0, and 4.0 min for HeLa cells, LPAAT3 MT cells, and LPAAT6 cells, respectively, and 11 min for LPAAT3 WT cells. (c) To measure LPAAT3 knockdown cells treated with BFA, the experiment above was repeated at 24°C where the t1/2 values for cells was 18 min for control cells, 25 min for cells overexpressing LPAAT3 WT, and 11.2 min for LPAAT3 knockdown cells. Data are representative of three independent experiments. (d) Control cells, LPAAT3 WT cells, LPAAT3 MT cells, and LPAAT3 siRNA knockdown cells were treated with 50 µM of the LPAT inhibitor CI-976 for 30 min. Cells expressing LPAAT3 WT were slower at forming Golgi membrane tubules, whereas knockdown cells were considerably faster. Bar = 20 µm. (e) Quantification of the CI-976 results. All error bars are SD for three independent experiments (n = 300).
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fig3: LPAAT3 overexpression slows BFA and CI-976–stimulated Golgi retrograde trafficking to the ER. (a) HeLa cells and cells transfected with LPAAT3 WT or LPAAT3 MT were treated with 10 µg/ml BFA and analyzed by immunofluorescence with GPP130 as a Golgi marker. Control cells, LPAAT3 MT cells, and LPAAT6 cells formed Golgi membrane tubules from 2 to 5 min after addition of BFA and were completely relocalized to ER membranes within 10 min, whereas LPAAT3 WT cells were slower. Bar = 20 µm. (b) The t1/2 of Golgi loss due to BFA tubulation was 5.5, 5.0, and 4.0 min for HeLa cells, LPAAT3 MT cells, and LPAAT6 cells, respectively, and 11 min for LPAAT3 WT cells. (c) To measure LPAAT3 knockdown cells treated with BFA, the experiment above was repeated at 24°C where the t1/2 values for cells was 18 min for control cells, 25 min for cells overexpressing LPAAT3 WT, and 11.2 min for LPAAT3 knockdown cells. Data are representative of three independent experiments. (d) Control cells, LPAAT3 WT cells, LPAAT3 MT cells, and LPAAT3 siRNA knockdown cells were treated with 50 µM of the LPAT inhibitor CI-976 for 30 min. Cells expressing LPAAT3 WT were slower at forming Golgi membrane tubules, whereas knockdown cells were considerably faster. Bar = 20 µm. (e) Quantification of the CI-976 results. All error bars are SD for three independent experiments (n = 300).
Mentions: Pharmacological studies have suggested that Golgi membrane tubules are positively influenced by PLA2 activity and negatively influenced by LPAT activity (Brown et al., 2003). Cells were all treated with the fungal metabolite brefeldin A (BFA), which generates numerous membrane tubules that redistribute Golgi membranes to the ER (Lippincott-Schwartz et al., 1989). Cells overexpressing LPAAT3 form Golgi tubules much slower than control and LPAAT3 MT cells (Fig. 3, a and b). Moreover, the Golgi tubules that did form in LPAAT3 WT overexpressing cells were far fewer in number and shorter in length than untransfected cells, as shown by Sholl analyses (Fig. S3, a and b; Sholl, 1953). The inhibition of BFA-stimulated tubule formation by LPAAT3 overexpression was not due to ineffective COPI disruption (Fig. S3 c). In contrast to overexpression, LPAAT3 knockdown cells exhibited a very rapid loss of the Golgi in BFA, so the experiment was repeated at 24°C to obtain quantitative data. The results showed that the half-time for loss of the Golgi in siRNA-treated cells was 11 min compared with 25 min for overexpressing cells (17 min in control cells) (Fig. 3 c).

Bottom Line: Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro.Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA).These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Recent studies have suggested that the functional organization of the Golgi complex is dependent on phospholipid remodeling enzymes. Here, we report the identification of an integral membrane lysophosphatidic acid-specific acyltransferase, LPAAT3, which regulates Golgi membrane tubule formation, trafficking, and structure by altering phospholipids and lysophospholipids. Overexpression of LPAAT3 significantly inhibited the formation of Golgi membrane tubules in vivo and in vitro. Anterograde and retrograde protein trafficking was slower in cells overexpressing LPAAT3 and accelerated in cells with reduced expression (by siRNA). Golgi morphology was also dependent on LPAAT3 because its knockdown caused the Golgi to become fragmented. These data are the first to show a direct role for a specific phospholipid acyltransferase in regulating membrane trafficking and organelle structure.

Show MeSH