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Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.

Lorente-Rodríguez A, Barlowe C - Mol. Biol. Cell (2010)

Bottom Line: The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport.Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors.Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.

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Screen for inhibitors of ER-to-Golgi transport. (A) Washed semi-intact cells containing [35S]gpαf were pretreated with indicated inhibitors for 20 min at 4°C. Transport reactions were then incubated with Recon proteins (COPII, Uso1p, and LMA1) and an ATP regeneration system at 23°C for 1 h. The amount of Golgi-modified [35S]gpαf was measured to determine transport efficiency. NA is the background level of transport in the absence of transport factors. (B) Semi-intact cell acceptor membranes were pretreated with indicated inhibitors for 20 min at 4°C and then incubated with COPII vesicles containing [35S]gpαf in the presence of fusion factors (U/L: Uso1p and LMA1) and an ATP regeneration system at 23°C for 1 h. Golgi-modified [35S]gpαf was measured to determine fusion efficiency. The no Acpt condition represents the background level of fusion in the absence of semi-intact cell acceptor membranes. (C) Pretreated, semi-intact cells as in panel A were incubated with COPII or COPII plus Uso1p for 30 min at 23°C. To measure budding (black bars) or tethering (white bars), diffusible vesicles containing [35S]gpαf were separated from semi-intact cell membranes by centrifugation. (D) Mock-budding reactions as in panel C were treated with trypsin, and total protease protected [35S]gpαf was quantified to assess membrane integrity.
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Figure 1: Screen for inhibitors of ER-to-Golgi transport. (A) Washed semi-intact cells containing [35S]gpαf were pretreated with indicated inhibitors for 20 min at 4°C. Transport reactions were then incubated with Recon proteins (COPII, Uso1p, and LMA1) and an ATP regeneration system at 23°C for 1 h. The amount of Golgi-modified [35S]gpαf was measured to determine transport efficiency. NA is the background level of transport in the absence of transport factors. (B) Semi-intact cell acceptor membranes were pretreated with indicated inhibitors for 20 min at 4°C and then incubated with COPII vesicles containing [35S]gpαf in the presence of fusion factors (U/L: Uso1p and LMA1) and an ATP regeneration system at 23°C for 1 h. Golgi-modified [35S]gpαf was measured to determine fusion efficiency. The no Acpt condition represents the background level of fusion in the absence of semi-intact cell acceptor membranes. (C) Pretreated, semi-intact cells as in panel A were incubated with COPII or COPII plus Uso1p for 30 min at 23°C. To measure budding (black bars) or tethering (white bars), diffusible vesicles containing [35S]gpαf were separated from semi-intact cell membranes by centrifugation. (D) Mock-budding reactions as in panel C were treated with trypsin, and total protease protected [35S]gpαf was quantified to assess membrane integrity.

Mentions: Inhibitors were initially screened for defects of overall ER-to-Golgi transport. In this assay, washed semi-intact cell membranes containing translocated [35S]glyco-pro-alpha-factor (gpαf) were pretreated with indicated inhibitors for 20 min at 4°C. Reactions were then incubated for 1 h at 23°C with purified transport factors to drive transport of gpαf to the Golgi complex (Barlowe, 1997). Upon delivery to the Golgi complex, gpαf receives outer-chain α1,6-mannose residues that can be immunoprecipitated with anti-1,6-mannose-specific serum to quantify gpαf transport (Baker et al., 1988). As observed in Figure 1A, U73122 and PH strongly inhibited ER-to-Golgi transport, whereas C1b, PI-PLC, and FYVE moderately inhibited the reaction. We next determined the specific stage(s) of ER-to-Golgi transport (budding, tethering, or fusion) that were affected by each inhibitor.FIGURE 1:


Requirement for Golgi-localized PI(4)P in fusion of COPII vesicles with Golgi compartments.

Lorente-Rodríguez A, Barlowe C - Mol. Biol. Cell (2010)

Screen for inhibitors of ER-to-Golgi transport. (A) Washed semi-intact cells containing [35S]gpαf were pretreated with indicated inhibitors for 20 min at 4°C. Transport reactions were then incubated with Recon proteins (COPII, Uso1p, and LMA1) and an ATP regeneration system at 23°C for 1 h. The amount of Golgi-modified [35S]gpαf was measured to determine transport efficiency. NA is the background level of transport in the absence of transport factors. (B) Semi-intact cell acceptor membranes were pretreated with indicated inhibitors for 20 min at 4°C and then incubated with COPII vesicles containing [35S]gpαf in the presence of fusion factors (U/L: Uso1p and LMA1) and an ATP regeneration system at 23°C for 1 h. Golgi-modified [35S]gpαf was measured to determine fusion efficiency. The no Acpt condition represents the background level of fusion in the absence of semi-intact cell acceptor membranes. (C) Pretreated, semi-intact cells as in panel A were incubated with COPII or COPII plus Uso1p for 30 min at 23°C. To measure budding (black bars) or tethering (white bars), diffusible vesicles containing [35S]gpαf were separated from semi-intact cell membranes by centrifugation. (D) Mock-budding reactions as in panel C were treated with trypsin, and total protease protected [35S]gpαf was quantified to assess membrane integrity.
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Related In: Results  -  Collection

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Figure 1: Screen for inhibitors of ER-to-Golgi transport. (A) Washed semi-intact cells containing [35S]gpαf were pretreated with indicated inhibitors for 20 min at 4°C. Transport reactions were then incubated with Recon proteins (COPII, Uso1p, and LMA1) and an ATP regeneration system at 23°C for 1 h. The amount of Golgi-modified [35S]gpαf was measured to determine transport efficiency. NA is the background level of transport in the absence of transport factors. (B) Semi-intact cell acceptor membranes were pretreated with indicated inhibitors for 20 min at 4°C and then incubated with COPII vesicles containing [35S]gpαf in the presence of fusion factors (U/L: Uso1p and LMA1) and an ATP regeneration system at 23°C for 1 h. Golgi-modified [35S]gpαf was measured to determine fusion efficiency. The no Acpt condition represents the background level of fusion in the absence of semi-intact cell acceptor membranes. (C) Pretreated, semi-intact cells as in panel A were incubated with COPII or COPII plus Uso1p for 30 min at 23°C. To measure budding (black bars) or tethering (white bars), diffusible vesicles containing [35S]gpαf were separated from semi-intact cell membranes by centrifugation. (D) Mock-budding reactions as in panel C were treated with trypsin, and total protease protected [35S]gpαf was quantified to assess membrane integrity.
Mentions: Inhibitors were initially screened for defects of overall ER-to-Golgi transport. In this assay, washed semi-intact cell membranes containing translocated [35S]glyco-pro-alpha-factor (gpαf) were pretreated with indicated inhibitors for 20 min at 4°C. Reactions were then incubated for 1 h at 23°C with purified transport factors to drive transport of gpαf to the Golgi complex (Barlowe, 1997). Upon delivery to the Golgi complex, gpαf receives outer-chain α1,6-mannose residues that can be immunoprecipitated with anti-1,6-mannose-specific serum to quantify gpαf transport (Baker et al., 1988). As observed in Figure 1A, U73122 and PH strongly inhibited ER-to-Golgi transport, whereas C1b, PI-PLC, and FYVE moderately inhibited the reaction. We next determined the specific stage(s) of ER-to-Golgi transport (budding, tethering, or fusion) that were affected by each inhibitor.FIGURE 1:

Bottom Line: The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport.Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors.Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.

ABSTRACT
The role of specific membrane lipids in transport between endoplasmic reticulum (ER) and Golgi compartments is poorly understood. Using cell-free assays that measure stages in ER-to-Golgi transport, we screened a variety of enzyme inhibitors, lipid-modifying enzymes, and lipid ligands to investigate requirements in yeast. The pleckstrin homology (PH) domain of human Fapp1, which binds phosphatidylinositol-4-phosphate (PI(4)P) specifically, was a strong and specific inhibitor of anterograde transport. Analysis of wild type and mutant PH domain proteins in addition to recombinant versions of the Sac1p phosphoinositide-phosphatase indicated that PI(4)P was required on Golgi membranes for fusion with coat protein complex II (COPII) vesicles. PI(4)P inhibition did not prevent vesicle tethering but significantly reduced formation of soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes between vesicle and Golgi SNARE proteins. Moreover, semi-intact cell membranes containing elevated levels of the ER-Golgi SNARE proteins and Sly1p were less sensitive to PI(4)P inhibitors. Finally, in vivo analyses of a pik1 mutant strain showed that inhibition of PI(4)P synthesis blocked anterograde transport from the ER to early Golgi compartments. Together, the data presented here indicate that PI(4)P is required for the SNARE-dependent fusion stage of COPII vesicles with the Golgi complex.

Show MeSH
Related in: MedlinePlus