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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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The Golgi fragments in LPAAT3 knockdown cells. (a) Cells stably overexpressing LPAAT3-GFP were transfected with a mixture of four siRNA molecules targeting LPAAT3 mRNA and analyzed by Western blot of cell lysates using anti-GFP and mannosidase II antibodies. (b) LPAAT3 expression was reduced by 85% (n = 3). (c) Immunofluorescence of GPP130 in LPAAT3 knockdown cells showed fragmented Golgi membranes. Bar = 20 µm. (d) The percentage of cells with fragmented Golgi ribbons as observed by immunofluorescence. (e) Golgi fragments contain markers for the cis-Golgi (10E6), the medial-Golgi (ManII) and the TGN (M6PR). Bar = 20 µm. (f) EM of thin sections from control, LPAAT3-GFP expressing, and knockdown cells. Higher magnification views of each boxed region are shown below. Bars = 500 nm.
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fig2: The Golgi fragments in LPAAT3 knockdown cells. (a) Cells stably overexpressing LPAAT3-GFP were transfected with a mixture of four siRNA molecules targeting LPAAT3 mRNA and analyzed by Western blot of cell lysates using anti-GFP and mannosidase II antibodies. (b) LPAAT3 expression was reduced by 85% (n = 3). (c) Immunofluorescence of GPP130 in LPAAT3 knockdown cells showed fragmented Golgi membranes. Bar = 20 µm. (d) The percentage of cells with fragmented Golgi ribbons as observed by immunofluorescence. (e) Golgi fragments contain markers for the cis-Golgi (10E6), the medial-Golgi (ManII) and the TGN (M6PR). Bar = 20 µm. (f) EM of thin sections from control, LPAAT3-GFP expressing, and knockdown cells. Higher magnification views of each boxed region are shown below. Bars = 500 nm.

Mentions: Cells stably expressing LPAAT3-GFP were used to examine Golgi morphology in siRNA knockdown experiments. Knockdown was verified by Western blot, which showed an average of 85% reduction in LPAAT3 expression (Fig. 2, a and b). In LPAAT3 knockdown cells, the Golgi complex was fragmented, forming numerous juxta-nuclear puncta (Fig. 2 c). Quantification showed that ∼90% of LPAAT3 knockdown cells had fragmented Golgi ribbons (Fig. 2 d). Each of the fragments colocalized with cis-Golgi markers (10E6), medial-Golgi markers (mannosidase II), and TGN markers (M6PR) (Fig. 2 e). Golgi fragmentation caused by LPAAT3 knockdown was reversed by transfection with an siRNA-resistant LPAAT3 construct (Fig. 2 d). Electron microscopy (EM) revealed that knockdown cells had multiple Golgi stacks throughout the cytoplasm (Fig. 2 f). The combined fluorescence and EM studies show that knockdown of LPAAT3 results in the formation of Golgi mini-stacks, which did not colocalize with the ER protein PDI, or the ER exit site marker Sec24 (unpublished data).


Lysophosphatidic acid acyltransferase 3 regulates Golgi complex structure and function.

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

The Golgi fragments in LPAAT3 knockdown cells. (a) Cells stably overexpressing LPAAT3-GFP were transfected with a mixture of four siRNA molecules targeting LPAAT3 mRNA and analyzed by Western blot of cell lysates using anti-GFP and mannosidase II antibodies. (b) LPAAT3 expression was reduced by 85% (n = 3). (c) Immunofluorescence of GPP130 in LPAAT3 knockdown cells showed fragmented Golgi membranes. Bar = 20 µm. (d) The percentage of cells with fragmented Golgi ribbons as observed by immunofluorescence. (e) Golgi fragments contain markers for the cis-Golgi (10E6), the medial-Golgi (ManII) and the TGN (M6PR). Bar = 20 µm. (f) EM of thin sections from control, LPAAT3-GFP expressing, and knockdown cells. Higher magnification views of each boxed region are shown below. Bars = 500 nm.
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fig2: The Golgi fragments in LPAAT3 knockdown cells. (a) Cells stably overexpressing LPAAT3-GFP were transfected with a mixture of four siRNA molecules targeting LPAAT3 mRNA and analyzed by Western blot of cell lysates using anti-GFP and mannosidase II antibodies. (b) LPAAT3 expression was reduced by 85% (n = 3). (c) Immunofluorescence of GPP130 in LPAAT3 knockdown cells showed fragmented Golgi membranes. Bar = 20 µm. (d) The percentage of cells with fragmented Golgi ribbons as observed by immunofluorescence. (e) Golgi fragments contain markers for the cis-Golgi (10E6), the medial-Golgi (ManII) and the TGN (M6PR). Bar = 20 µm. (f) EM of thin sections from control, LPAAT3-GFP expressing, and knockdown cells. Higher magnification views of each boxed region are shown below. Bars = 500 nm.
Mentions: Cells stably expressing LPAAT3-GFP were used to examine Golgi morphology in siRNA knockdown experiments. Knockdown was verified by Western blot, which showed an average of 85% reduction in LPAAT3 expression (Fig. 2, a and b). In LPAAT3 knockdown cells, the Golgi complex was fragmented, forming numerous juxta-nuclear puncta (Fig. 2 c). Quantification showed that ∼90% of LPAAT3 knockdown cells had fragmented Golgi ribbons (Fig. 2 d). Each of the fragments colocalized with cis-Golgi markers (10E6), medial-Golgi markers (mannosidase II), and TGN markers (M6PR) (Fig. 2 e). Golgi fragmentation caused by LPAAT3 knockdown was reversed by transfection with an siRNA-resistant LPAAT3 construct (Fig. 2 d). Electron microscopy (EM) revealed that knockdown cells had multiple Golgi stacks throughout the cytoplasm (Fig. 2 f). The combined fluorescence and EM studies show that knockdown of LPAAT3 results in the formation of Golgi mini-stacks, which did not colocalize with the ER protein PDI, or the ER exit site marker Sec24 (unpublished data).

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