1887

Abstract

Phospholipids in cells are mainly synthesized in the cytoplasmic leaflet of the endoplasmic reticulum (ER) and the newly made polar lipids must flip-flop rapidly across biological membranes to sustain cellular life. But this 'flipping' is energetically costly as well as its translocation rate is low. As a solution to this, cells have membrane proteins that function as lipid transporters - 'flippases', proteins that facilitates the rapid, bi-directional, energy-independent flip-flop of phospholipids between the cytosolic and lumenal leaflets of the ER membrane. Flippases have been known to play a key role in membrane stability as well as in the mechanism by which cells avoid being killed by macrophages. Candidate flippases have been implicated in human diseases that include intrahepatic cholestasis, angelman syndrome, autism, tangier disease, macular dystrophy and adrenoleukodystrophy. Establishing the primary function of candidate flippases and how they contribute to cell function and human disease is becoming a central issue in biology. Although the flippases that operate at the plasma membrane of eukaryotes at the expense of ATP hydrolysis resulting in unidirectional lipid flipping have been identified, the ATP-independent bi-directional flippases that translocate lipids in specialized compartments such as the ER have not yet been identified at their molecular level. The objective of the current study is to identify ER flippases in yeast Saccharomyces cerevisiae using a quantitative proteomics approach based on stable isotope labeling by amino acids in cell culture (SILAC). Yeast cells were grown in synthetic medium supplemented with either 'light' or 'heavy' lysine. Proteins extracted from unlabeled (light) cells were further fractionated by velocity sedimentation in a glycerol gradient and flippase activity of each fraction was quantified by a phospholipid reconstitution-based procedure. An aliquot of labeled (heavy) extract (containing equal amount of protein by weight) was added to each light fraction. The mixed fractions were then subjected to in-gel digestion followed by quantitative proteomic analysis using mass spectrometry. The data obtained were processed using MaxQuant followed by Spearman correlation analysis for identification of proteins with enrichment profiles matching that of the activity profile. The potential flippase candidates were tested for their activity using genomically tagged yeast strains.

Loading

Article metrics loading...

/content/papers/10.5339/qfarf.2013.BIOP-0180
2013-11-20
2020-04-07
Loading full text...

Full text loading...

http://instance.metastore.ingenta.com/content/papers/10.5339/qfarf.2013.BIOP-0180
Loading
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error