Quantitative Profiling of the Membrane Proteome in a Halophilic Archaeon

Abstract

We present a large scale quantitation study of the membrane proteome from Halobacterium salinarum. To overcome problems generally encountered with membrane proteins, we established a membrane preparation protocol that allows the application of most proteomic techniques originally developed for soluble proteins. Proteins were quantified using two complementary approaches. For gel-based quantitation, DIGE labeling was combined with two-dimensional gel electrophoresis on an improved 16-benzyldimethyl-n-hexadecylammonium chloride/SDS system. MS-based quantitation was carried out by combining gel-free separation with the recently developed isotope-coded protein labeling technique. Good correlations between these two independent quantitation strategies were obtained. From computational analysis we conclude that labeling of free amino groups by isotopecoded protein labeling (Lys and free N termini) is better suited for membrane proteins than Cys-based labeling strategies but that quantitation of integral membrane proteins remains cumbersome compared with soluble proteins. Nevertheless we could quantify 155 membrane proteins; 101 of these had transmembrane domains. We compared two growth states that strongly affect the energy supply of the cells: aerobic versus anaerobic/phototrophic conditions. The photosynthetic protein bacteriorhodopsin is the most highly regulated protein. As expected, several other membrane proteins involved in aerobic or anaerobic energy metabolism were found to be regulated, but in total, however, the number of regulated proteins is rather small. Molecular & Cellular Proteomics 5:1543-1558, 2006.

Consequences of Membrane Protein Overexpression in Escherichia coli

“…Furthermore the levels of membrane-associated ribosomes were increased upon membrane protein overexpression, whereas total levels of ribosomes in the cells did not change. This is in keeping with previous E. coli studies that showed that high rates of membrane protein translation result in more membrane-associated ribosomes (61,62). Our data suggest that upon membrane protein overexpression most Sec translocons are engaged in co-translational protein translocation, i.e.…”

Towards Glycoengineering in Archaea: Replacement of Haloferax volcanii AglD with Homologous Glycosyltransferases from Other Halophilic Archaea

“…Moreover, OE1482 was not detected when the membrane proteomes of Hbt. salinarum cells grown under the same anaerobic and phototrophic or aerobic conditions were addressed (6). How thus can the presence of a seemingly nontranscribed glycosyltransferaseencoding gene in the Hbt.…”

A Multiplexed Quantitative Strategy for Membrane Proteomics

“…In a recent study, Bisle et al (38) reported protein-level labeling on lysine and the N terminus (isotopecoded protein labeling) of integral membrane proteins and compared the efficiency with ICAT (labeling cysteine). Although the isotope-coded protein labeling strategy labels lysines, which are more common than cysteines, the technical difficulty was still apparent in the experimental observation that there was, on average, only a single detectable lysine-containing peptide per integral membrane protein.…”

“…Fig. 7 summarizes some of these proteins that have been confirmed to be involved in the major abnormalities in epithelial cell proliferation and apoptosis, cell-cell and cell-matrix interactions, abnormal ion and fluid secretion, and alterations in membrane protein polarity (38).…”