Lipid rafts, microdomain heterogeneity and inter‐organelle contacts: Impacts on membrane preparation for proteomic studies

Abstract

In recent years, there has been considerable interest in mapping the protein content of isolated organelles using mass spectrometry. However, many subcellular compartments are highly dynamic with diverse and intricate architectures that are not always preserved during membrane isolation procedures. Furthermore, lateral heterogeneities in intra-membrane lipid and protein concentrations underlie the formation of membrane microdomains, trafficking vesicles and inter-membrane contacts. These complexities in membrane organisation have important consequences for the design of membrane preparation strategies and test the very concept of organelle purity. We illustrate how some of these biological considerations are relevant to membrane preparation and assess the numerous potential pitfalls in attempting to purify organelles from mammalian cells.

Peripheral Myelin Protein 22 Preferentially Partitions into Ordered Phase Membrane Domains

“…While there are a number of biochemical reports that identify membrane proteins founds to colocalize with biochemically isolated "lipid raft" cell fractions (53)(54)(55), to the best of our knowledge this is the first work that quantitively demonstrates the ordered phase membrane domain preference of a multi-pass membrane protein in cell-derived GPMVs.…”

Raft-like membranes from the trans-Golgi network and endosomal compartments

“…The development of this protocol was highly influenced by earlier work aimed at purifying detergent-or carbonate-resistant membrane microdomains from the plasma membrane 54,55,73,74 . These early experiments were controversial in terms of their interpretation and use, but they nevertheless laid the foundations for many current ideas on membrane organization and, in particular, the lipid raft hypothesis 13,[75][76][77][78] .…”

“…Furthermore, although the removal of peripheral membrane proteins can result in a membrane fraction becoming less dense and more raft-like 72 , these biophysical properties alone are not sufficient to infer that the buoyant fraction consists solely of purified lipid rafts from the TGN. As with all subcellular fraction protocols 73 , it is possible that membrane fragments from other cellular compartments are present to some degree in the isolated fraction. More specifically in this case, it is possible that carbonate-stripped membrane fragments from the Golgi or early endosome compartments could cofractionate with the buoyant fraction.…”

Interaction of membrane/lipid rafts with the cytoskeleton: Impact on signaling and function

“…As a result, one can isolate MLR based on detergent-resistance and subsequent fractionation on density gradients[31, 32]. Early experiments isolated MLR with 1% Triton X-100 followed by sucrose density gradient fractionation[31]; later studies employed other detergents including Lubrol, Brij, Nonidet, CHAPS, and octylglucoside at varying concentrations[14, 33]. However, concerns arose that use of such detergents may produce a `false' clustering of raft lipids with proteins, interactions that do not exist in cells[33].…”

“…Lysis buffers that do not sufficiently disrupt the cytoskeleton to properly separate different species of MLR (non-caveolar versus caveolar) can also produce confounding results, especially when performed on cells highly abundant in cytoskeletal architeture, such as adult cardiac myocytes[36, 42]. Although many types of preparations have contributed to current understanding of MLR structure and function, their limitations provide a rationale for the development of improved methodologies that optimize membrane purification and enrichment strategies[33]. For example, improved fractionation methods in combination with morphological approaches, such as super-resolution microscopy that allows for fluorescence imaging with a precision near 20 nm, may prove useful for obtaining more consistent and accurate understanding of MLR organization and function[43].…”