Targeting of the yeast phosphatidylserine transporter Osh6 to membrane contact sites

JUAN MARTÍN D'AMBROSIO; VÉRONIQUE ALBANÈSE ; ALENKA COPIC

Ljubljana

Congreso; 4th Yeast Lipid Conference YLC 2019; 2019

The "Yeast Lipid Conference"

Difference in lipid composition between thecompartments of eukaryotic cells is maintained by lipid transfer proteins (LTP).In budding yeast, the LTP Osh6 and Osh7, which are members of theoxysterol-binding protein family, localise to the cytosol and to the membranecontact sites (MCS) between the endoplasmic reticulum (ER) and the plasmamembrane (PM).  There, they mediate thetransport of phosphatidylserine (PS), which is synthesized at the ER, inexchange for phosphatidylinositol-4-phosphate (PI4P). Sequence and structuralanalyses showed that Osh6 and Osh7 contain a single domain that canalternatively carry PS or PI4P, but offer no clues about how these proteins couldlocalize to the MCS. We have searched for MCS targeted proteins that couldanchor Osh6 and Osh7, using the split-venus assay and the yeast two-hybridsystem to test for protein interactions. We have also generated Osh6 mutants thatare misslocalized in the cell, and we analyze how these mutations affect PStransport to the PM. We use amicrofluidics-based fluorescent assay to follow the transport ofnewly-synthesized PS in yeast in real time, by measuring the distribution ofthe PS reporter C2Lact-GFP. We take advantage of cells lacking Cho1,enzyme responsible for PS synthesis in yeast, to which lyso-PS can be addedexogenously and is taken up and converted to PS at the ER. These experiments address the question how protein targeting affects PS transportin yeast.<!-- /* Font Definitions */ @font-face{font-family:"Cambria Math";panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:3 0 0 0 1 0;}@font-face{font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;mso-font-charset:0;mso-generic-font-family:swiss;mso-font-pitch:variable;mso-font-signature:-536859905 -1073732485 9 0 511 0;}@font-face{font-family:Cambria;panose-1:2 4 5 3 5 4 6 3 2 4;mso-font-charset:0;mso-generic-font-family:roman;mso-font-pitch:variable;mso-font-signature:-536870145 1073743103 0 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal{mso-style-unhide:no;mso-style-qformat:yes;mso-style-parent:"";margin:0cm;margin-bottom:.0001pt;mso-pagination:widow-orphan;font-size:12.0pt;font-family:"Cambria",serif;mso-fareast-font-family:Cambria;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US;}p.Abstract, li.Abstract, div.Abstract{mso-style-name:Abstract;mso-style-unhide:no;margin-top:18.0pt;margin-right:14.4pt;margin-bottom:0cm;margin-left:14.4pt;margin-bottom:.0001pt;text-align:justify;mso-pagination:widow-orphan;font-size:10.0pt;font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US;}.MsoChpDefault{mso-style-type:export-only;mso-default-props:yes;font-size:10.0pt;mso-ansi-font-size:10.0pt;mso-bidi-font-size:10.0pt;font-family:"Cambria",serif;mso-ascii-font-family:Cambria;mso-fareast-font-family:Cambria;mso-hansi-font-family:Cambria;mso-ansi-language:NL-BE;mso-fareast-language:NL-BE;}size:612.0pt 792.0pt;margin:70.85pt 70.85pt 70.85pt 70.85pt;mso-header-margin:36.0pt;mso-footer-margin:36.0pt;mso-paper-source:0;}div.WordSection1{page:WordSection1;}