Limits...
WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion.

King JS, Gueho A, Hagedorn M, Gopaldass N, Leuba F, Soldati T, Insall RH - Mol. Biol. Cell (2013)

Bottom Line: Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed.Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH- lysosomes.This causes the formation of multilamellar bodies typical of many lysosomal storage diseases.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, United Kingdom.

ABSTRACT
Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH- lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused.

Show MeSH

Related in: MedlinePlus

Proteomic analysis of mutant phagosomes. (A) The relative proportion of proteins from different compartments is reduced by at least 30% in both WASH and FAM21 mutant phagosomes. Eighty-five proteins in all were identified by this criteria. (B) Identity and relative abundance of lysosomal and secreted (below dashed line) proteins identified in this screen. Lysosomal proteins only identified in one mutant are also listed. The relative abundance of v-ATPase subunits is included in the lower section. *, p < 0.05; **, p < 0.005; ***, p < 0.001 (Student's t-test).
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3756923&req=5

Figure 3: Proteomic analysis of mutant phagosomes. (A) The relative proportion of proteins from different compartments is reduced by at least 30% in both WASH and FAM21 mutant phagosomes. Eighty-five proteins in all were identified by this criteria. (B) Identity and relative abundance of lysosomal and secreted (below dashed line) proteins identified in this screen. Lysosomal proteins only identified in one mutant are also listed. The relative abundance of v-ATPase subunits is included in the lower section. *, p < 0.05; **, p < 0.005; ***, p < 0.001 (Student's t-test).

Mentions: Phagosome maturation is a highly regulated and organized process during which lysosomal components are sequentially delivered and retrieved (Clarke et al., 2002; Gotthardt et al., 2002, 2006a). To establish the mechanism underlying the defects in degradation, we used quantitative comparative proteomics to identify differentially represented proteins in the isolated phagosomes of Ax2, WASH, and FAM21 mutants (Dieckmann et al., 2012; Gotthardt et al., 2006a). To confirm this approach, we first looked at the levels of v-ATPase. In Dictyostelium, WASH is required for v-ATPase recycling, and WASH mutants retain v-ATPase on their endosomes (Carnell et al., 2011). Consistent with this, all nine subunits of the v-ATPase complex were elevated in the WASH- phagocytic compartment compared with wild-type (Figure 3B). In contrast, FAM21­ mutants retain WASH activity (Park et al., 2013), and several of the v-ATPase subunits were identified as decreased in FAM21- phagosomes relative to Ax2. This validates the data set and confirms that we are able to identify differentially trafficked proteins with this method.


WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion.

King JS, Gueho A, Hagedorn M, Gopaldass N, Leuba F, Soldati T, Insall RH - Mol. Biol. Cell (2013)

Proteomic analysis of mutant phagosomes. (A) The relative proportion of proteins from different compartments is reduced by at least 30% in both WASH and FAM21 mutant phagosomes. Eighty-five proteins in all were identified by this criteria. (B) Identity and relative abundance of lysosomal and secreted (below dashed line) proteins identified in this screen. Lysosomal proteins only identified in one mutant are also listed. The relative abundance of v-ATPase subunits is included in the lower section. *, p < 0.05; **, p < 0.005; ***, p < 0.001 (Student's t-test).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756923&req=5

Figure 3: Proteomic analysis of mutant phagosomes. (A) The relative proportion of proteins from different compartments is reduced by at least 30% in both WASH and FAM21 mutant phagosomes. Eighty-five proteins in all were identified by this criteria. (B) Identity and relative abundance of lysosomal and secreted (below dashed line) proteins identified in this screen. Lysosomal proteins only identified in one mutant are also listed. The relative abundance of v-ATPase subunits is included in the lower section. *, p < 0.05; **, p < 0.005; ***, p < 0.001 (Student's t-test).
Mentions: Phagosome maturation is a highly regulated and organized process during which lysosomal components are sequentially delivered and retrieved (Clarke et al., 2002; Gotthardt et al., 2002, 2006a). To establish the mechanism underlying the defects in degradation, we used quantitative comparative proteomics to identify differentially represented proteins in the isolated phagosomes of Ax2, WASH, and FAM21 mutants (Dieckmann et al., 2012; Gotthardt et al., 2006a). To confirm this approach, we first looked at the levels of v-ATPase. In Dictyostelium, WASH is required for v-ATPase recycling, and WASH mutants retain v-ATPase on their endosomes (Carnell et al., 2011). Consistent with this, all nine subunits of the v-ATPase complex were elevated in the WASH- phagocytic compartment compared with wild-type (Figure 3B). In contrast, FAM21­ mutants retain WASH activity (Park et al., 2013), and several of the v-ATPase subunits were identified as decreased in FAM21- phagosomes relative to Ax2. This validates the data set and confirms that we are able to identify differentially trafficked proteins with this method.

Bottom Line: Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed.Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH- lysosomes.This causes the formation of multilamellar bodies typical of many lysosomal storage diseases.

View Article: PubMed Central - PubMed

Affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, United Kingdom.

ABSTRACT
Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starvation. WASH is required for efficient phagosomal proteolysis, and proteomic analysis demonstrates that this is due to reduced delivery of lysosomal hydrolases. Both protease and lipase delivery are disrupted, and lipid catabolism is also perturbed. Starvation-induced autophagy therefore leads to phospholipid accumulation within WASH- lysosomes. This causes the formation of multilamellar bodies typical of many lysosomal storage diseases. Mechanistically, we show that, in cells lacking WASH, cathepsin D becomes trapped in a late endosomal compartment, unable to be recycled to nascent phagosomes and autophagosomes. WASH is therefore required for the maturation of lysosomes to a stage at which hydrolases can be retrieved and reused.

Show MeSH
Related in: MedlinePlus