Abstract
Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).
Highlights
All living cells define their boundaries by membranes containing multispan membrane proteins (MSPs), which often serve as selective pores or active pumps for nutrients and ions or as enzymes, for example, to produce membrane lipids
We were motivated by recent data indicating that the active site of numerous enzymes involved in lipid biosynthesis, e.g. of the membrane bound O-acyltransferases (MBOATs), probably resides in the ER lumen, but these claims need confirmation [1,2,3]
We estimated that a carbonate wash
Summary
All living cells define their boundaries by membranes containing multispan membrane proteins (MSPs), which often serve as selective pores or active pumps for nutrients and ions or as enzymes, for example, to produce membrane lipids. The elucidation of the 3D structure of such MSPs is work intensive as it typically requires the crystallization and X-ray diffraction of purified proteins maintained in solution by a belt of micellar detergent. We were motivated by recent data indicating that the active site of numerous enzymes involved in lipid biosynthesis, e.g. of the membrane bound O-acyltransferases (MBOATs), probably resides in the ER lumen, but these claims need confirmation [1,2,3]. Localizing the active site of such enzymes is of particular interest in that it suggests the existence of membrane transporters for substrates and products that may have to cross the membrane.
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