The yeast cell wall integrity pathway signals from recycling endosomes upon elimination of phosphatidylinositol (4,5)-bisphosphate by mammalian phosphatidylinositol 3-kinase.
Phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] is essential for recognition of the plasma membrane inner leaf by protein complexes. We expressed mammalian class I phosphoinositide 3-kinase (PI3K) in Saccharomyces cerevisiae to eliminate PtdIns(4,5)P(2) by its conversion into PtdIns(3,4,5)P(3), a lipid naturally missing in this yeast. This led to loss of actin function and endocytosis defects, causing a blockage in polarized secretion. Also, the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway was activated, triggering a typical transcriptional response. In the absence of PtdIns(4,5)P(2) at the plasma membrane, the Pkc1 protein kinase upstream the CWI MAPK module localized to post-Golgi endosomes marked by SNARE Snc1 and Rab GTPases Ypt31 and Ypt32. Other components at the head of the pathway, like the mechanosensor Wsc1, the GTPase Rho1 and its activator the GDP/GTP exchange factor Rom2, co-localized with Pkc1 in these compartments. Chemical inhibition of PI3K proved that both CWI activation and Pkc1 relocation to endosomes are reversible. These results suggest that the CWI pathway is able to respond to loss of plasma membrane identity from recycling endosomes.
Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain.
Teresa
T
Fernández-Acero
Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain.
Isabel
I
Rodríguez-Escudero
Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain. Electronic address: molmifa@ucm.es.
María
M
Molina
Dpto. de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; Instituto Ramón y Cajal de Investigaciones Sanitarias (IRYCIS), Madrid, Spain.
Víctor J
VJ
Cid
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D004705:Endocytosis / Q000235:genetics; D011992:Endosomes / Q000378:metabolism; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008565:Membrane Proteins / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000378:metabolism; D050683:R-SNARE Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D000081082:Phosphoinositide-3 Kinase Inhibitors; D050683:R-SNARE Proteins; C490929:ROM2 protein, S cerevisiae; C119383:SLG1 protein, S cerevisiae; C083241:SNC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D058539:Phosphatidylinositol 3-Kinase; C490702:PKC1 protein, S cerevisiae; D011493:Protein Kinase C; C087905:YPT31 protein, S cerevisiae; C121633:YPT32 protein, S cerevisiae; C051302:RHO1 protein, S cerevisiae; D020691:rab GTP-Binding Proteins; D020741:rho GTP-Binding Proteins
Endosomes; MAPK signaling; PI3K; Phosphoinositides; Pkc1; Yeast