Massive Formation of Intracellular Membrane Vesicles in Escherichia coli by a Monotopic Membrane-bound Lipid Glycosyltransferase

Abstract

The morphology and curvature of biological bilayers are determined by the packing shapes and interactions of their participant molecules. Bacteria, except photosynthetic groups, usually lack intracellular membrane organelles. Strong overexpression in Escherichia coli of a foreign monotopic glycosyltransferase (named monoglycosyldiacylglycerol synthase), synthesizing a nonbilayer-prone glucolipid, induced massive formation of membrane vesicles in the cytoplasm. Vesicle assemblies were visualized in cytoplasmic zones by fluorescence microscopy. These have a very low buoyant density, substantially different from inner membranes, with a lipid content of >60% (w/w). Cryo-transmission electron microscopy revealed cells to be filled with membrane vesicles of various sizes and shapes, which when released were mostly spherical (diameter ≈100 nm). The protein repertoire was similar in vesicle and inner membranes and dominated by the glycosyltransferase. Membrane polar lipid composition was similar too, including the foreign glucolipid. A related glycosyltransferase and an inactive monoglycosyldiacylglycerol synthase mutant also yielded membrane vesicles, but without glucolipid synthesis, strongly indicating that vesiculation is induced by the protein itself. The high capacity for membrane vesicle formation seems inherent in the glycosyltransferase structure, and it depends on the following: (i) lateral expansion of the inner monolayer by interface binding of many molecules; (ii) membrane expansion through stimulation of phospholipid synthesis, by electrostatic binding and sequestration of anionic lipids; (iii) bilayer bending by the packing shape of excess nonbilayer-prone phospholipid or glucolipid; and (iv) potentially also the shape or penetration profile of the glycosyltransferase binding surface. These features seem to apply to several other proteins able to achieve an analogous membrane expansion.

Membrane Invaginations in Bacteria and Mitochondria: Common Features and Evolutionary Scenarios

“…The morphology of the ICMs can range from membrane vesicles [Eriksson et al, 2009] to tubular membranes and stacks [Arechaga et al, 2000;Lefman et al, 2004]. This morphology could be determined by a number of factors, such as the size and shape of the overproduced protein, the charge of both protein and interacting lipids, the dynamic of these interactions, and the packing pressure exerted by the insertion of the overexpressed protein into the plasma membrane.…”

“…Thus, the overexpression of endogenous membrane proteins like the fumarate reductase [Weiner et al, 1984], the manitol permease MtlA [van Weeghel et al, 1990], the glycerol acyl transferase PlsB [Wilkison et al, 1986], the chemotaxis receptor Tsr [Lefman et al, 2004;Weis et al, 2003], the FtsY-receptor-ribosome complex [Herskovits et al, 2002], the ATP synthase complex [von Meyenburg et al, 1984] or the ATP synthase b subunit [Arechaga et al, 2000], induces the proliferation of membrane invaginations in the cytoplasm. Intracellular membranes (ICMs) have also been observed upon the heterologous overexpression in E. coli of the lipid glyscolyltransferases alMGS and alDGS from Acholeplasma laidawii [Eriksson et al, 2009], the alkane hydrolase AlkB from Pseudomonas oleovorans [Nieboer et al, 1996], and some viral proteins like the foot and mouth disease 3A gene product [Weber et al, 1996] or the bacteriophage PM2 sps6.6 gene [Armour and Brewer, 1990]. …”

“…Occasionally, large amounts of recombinant membrane proteins can be recovered from the membrane fraction, suggesting that they have been properly folded and inserted into the membrane. However, the bacterial inner membrane is already saturated with membrane protein complexes and there is not enough space to accommodate such an amount of recombinant protein, which could account for up to 20% of the total protein content of the cell [Arechaga et al, 2000;Eriksson et al, 2009]. Hence, the surface available for integration of the new proteins must be increased.…”

Membrane proteins, magic-angle spinning, and in-cell NMR

“…Under most conditions used for membrane protein overexpression, E. coli increases lipid synthesis to maintain a constant lipid-to-protein ratio (19). The proliferation of intracellular membrane and the synthesis of excess non-bilayer-prone lipids on overexpression have been reported (20)(21)(22). Perhaps the biggest obstacle is moving from bacterial cells to higher eukaryotic cells, where cell viability is harder to maintain and overexpression is more difficult.…”

Building Spatial Synthetic Biology with Compartments, Scaffolds, and Communities

“…2). This was accomplished by overexpressing foreign bacterial glucosyltransferases (Eriksson et al 2009) or by expressing eukaryotic calveolin (Walser et al 2012). The latter strategy produces morpholog- (Zhang et al 2006a).…”