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A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

Bozaquel-Morais BL, Madeira JB, Maya-Monteiro CM, Masuda CA, Montero-Lomeli M - PLoS ONE (2010)

Bottom Line: Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains.We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity.In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

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Statistical analysis of a high-throughput experiment employing the LFR assay.LDs were quantified by the LFR assay. The frequency distribution of the relative LD index of strains from the lipid synthesis group (A, n = 31) and protein phosphatase group (B, n = 65) was calculated. Data were collected from four independent experiments as described in the Materials and Methods section. The D'Agostino & Pearson normality test was employed at P<0.05. The lipid synthesis group was normally distributed (p = 0.281), while the protein phosphatase group failed to pass the normality test (p = 0.016). The arbitrary cohort (±20% in relation to WT values) is indicated in the graphs by a dashed line (llc, low lipid droplet content and hlc, high lipid droplet content).
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pone-0013692-g005: Statistical analysis of a high-throughput experiment employing the LFR assay.LDs were quantified by the LFR assay. The frequency distribution of the relative LD index of strains from the lipid synthesis group (A, n = 31) and protein phosphatase group (B, n = 65) was calculated. Data were collected from four independent experiments as described in the Materials and Methods section. The D'Agostino & Pearson normality test was employed at P<0.05. The lipid synthesis group was normally distributed (p = 0.281), while the protein phosphatase group failed to pass the normality test (p = 0.016). The arbitrary cohort (±20% in relation to WT values) is indicated in the graphs by a dashed line (llc, low lipid droplet content and hlc, high lipid droplet content).

Mentions: We employed the LFR assay to study the involvement of protein phosphatases in LD metabolism. In this high-throughput experiment, we screened 96 strains as well as the BY4741 WT strain (14 independent WT clones). We subdivided the strains in two groups. The first was a reference group, referred to as the lipid synthesis group. This reference group included 31 strains for genes encoding LD-associated enzymes and/or enzymes involved in the metabolism of sterols, the metabolism of triglycerides and the biosynthesis of glycerolipids (Table 1) [41]. The second group, referred to as the phosphatase group, was composed of 65 strains with all known non-essential protein phosphatases and non-essential phosphatase regulatory subunits (Table 2), according to the annotation in the Yeast Genome Database (www.yeastgenome.org, October 2009). As LD content varies substantially during growth (see Figure 4B) and not all strains have the same growth rate, we decided to asses LD content in strains grown to stationary phase, which was attained for all strains after 48 hours of seeding. A histogram of the distribution of the lipid indexes for all strains was analyzed and an index for cataloguing strains (as containing a higher LD index (hlc) or a lower LD index (llc)) was elaborated using an arbitrary cohort of ±20% in relation to the LD index of WT strains (Figure 5). When the mean LD index of the lipid synthesis group was compared to that of the WT strain, no significant difference was observed (means were 0.9539 and 0.9135, respectively p = 0.276). The same trend was not observed for the mean LD index of the protein phosphatase group, which was significantly higher than the WT sample (1.023, p = 0.0164) and included an over-representation of hlc strains. Interestingly, this result indicates that lipid accumulation is more likely provoked by the random loss of protein phosphatase function as opposed to the random loss of function among proteins involved in lipid metabolism. After analyzing the groups as a whole, individual strains were catalogued as having higher (hlc) or lower LD content (llc) (Tables 1 and 2).


A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

Bozaquel-Morais BL, Madeira JB, Maya-Monteiro CM, Masuda CA, Montero-Lomeli M - PLoS ONE (2010)

Statistical analysis of a high-throughput experiment employing the LFR assay.LDs were quantified by the LFR assay. The frequency distribution of the relative LD index of strains from the lipid synthesis group (A, n = 31) and protein phosphatase group (B, n = 65) was calculated. Data were collected from four independent experiments as described in the Materials and Methods section. The D'Agostino & Pearson normality test was employed at P<0.05. The lipid synthesis group was normally distributed (p = 0.281), while the protein phosphatase group failed to pass the normality test (p = 0.016). The arbitrary cohort (±20% in relation to WT values) is indicated in the graphs by a dashed line (llc, low lipid droplet content and hlc, high lipid droplet content).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965658&req=5

pone-0013692-g005: Statistical analysis of a high-throughput experiment employing the LFR assay.LDs were quantified by the LFR assay. The frequency distribution of the relative LD index of strains from the lipid synthesis group (A, n = 31) and protein phosphatase group (B, n = 65) was calculated. Data were collected from four independent experiments as described in the Materials and Methods section. The D'Agostino & Pearson normality test was employed at P<0.05. The lipid synthesis group was normally distributed (p = 0.281), while the protein phosphatase group failed to pass the normality test (p = 0.016). The arbitrary cohort (±20% in relation to WT values) is indicated in the graphs by a dashed line (llc, low lipid droplet content and hlc, high lipid droplet content).
Mentions: We employed the LFR assay to study the involvement of protein phosphatases in LD metabolism. In this high-throughput experiment, we screened 96 strains as well as the BY4741 WT strain (14 independent WT clones). We subdivided the strains in two groups. The first was a reference group, referred to as the lipid synthesis group. This reference group included 31 strains for genes encoding LD-associated enzymes and/or enzymes involved in the metabolism of sterols, the metabolism of triglycerides and the biosynthesis of glycerolipids (Table 1) [41]. The second group, referred to as the phosphatase group, was composed of 65 strains with all known non-essential protein phosphatases and non-essential phosphatase regulatory subunits (Table 2), according to the annotation in the Yeast Genome Database (www.yeastgenome.org, October 2009). As LD content varies substantially during growth (see Figure 4B) and not all strains have the same growth rate, we decided to asses LD content in strains grown to stationary phase, which was attained for all strains after 48 hours of seeding. A histogram of the distribution of the lipid indexes for all strains was analyzed and an index for cataloguing strains (as containing a higher LD index (hlc) or a lower LD index (llc)) was elaborated using an arbitrary cohort of ±20% in relation to the LD index of WT strains (Figure 5). When the mean LD index of the lipid synthesis group was compared to that of the WT strain, no significant difference was observed (means were 0.9539 and 0.9135, respectively p = 0.276). The same trend was not observed for the mean LD index of the protein phosphatase group, which was significantly higher than the WT sample (1.023, p = 0.0164) and included an over-representation of hlc strains. Interestingly, this result indicates that lipid accumulation is more likely provoked by the random loss of protein phosphatase function as opposed to the random loss of function among proteins involved in lipid metabolism. After analyzing the groups as a whole, individual strains were catalogued as having higher (hlc) or lower LD content (llc) (Tables 1 and 2).

Bottom Line: Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains.We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity.In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Bioquímica Médica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

Show MeSH