1. THE MODEL SYSTEM: PREPARATION AND CHARACTERIZATION OF BACTERIAL MEMBRANE VESICLES H.R. Kaback

2. Figure 1. Electron micrograph of an intact Escherichia coli cell
Figure 1. Electron micrograph of an intact Escherichia coli cell. LPs, lipopolysaccharide or outer membrane which is present in Gram-negative bacteria and contains various porins which allow diffusion of small molecules (up to penta-lysine) into the so-called peri-plasmic space. Not seen in the micrograph is the rigid, electron dense peptidoglycan layer of the cell wall, present in all Eubactericiae which provides the cell with its shape and allows bacteria to accumulate metabolites again large concentration gradients without bursting due to influx of water. Closely opposed to the peptidoglycan layer is the plasma membrane (PM).

3. Figure 2. Spheroplasts. Treatment of E
Figure 2. Spheroplasts. Treatment of E. coli with lysozyme which degrades the peptidoglycan layer in the presence of ethylene-diametetraacetate (EDTA) to permeabilize the outer membrane leads to the formation of osmotically fragile cells (spheroplasts) that are stable under hypertonic conditions. (i.e., 20-30% sucrose). Alternatively, cells can be grown in the presence of penicillin which inhibits peptidoglycan synthesis

4. Figure 3. Formation of membrane vesicles by osmotic lysis involves membrane rupture and resealing (i.e., the membrane has the properties of a phospholipid bilayer): Top. Addition of colloidal gold (visible in the electron microscope) to spheroplasts reveals gold particles outside, none within. Middle. Same with membrane vesicles. Bottom. Osmotic lysis of spheroplasts in the presence of colloidal gold leads to vesicles with colloidal gold trapped inside. Therefore, release of intracellular contents during lysis involves rupture of the plasma membrane and resealing.
During lysis, the intracellular space equilibrates with the external medium. Thus, in addition to emptying the cell of its soluble contents, during lysis, large molecule such as enzymes or antibodies can be introduced into the intravesicular space.

5. Figure 4. Electron micrograph of membrane vesicles
Figure 4. Electron micrograph of membrane vesicles. Vesicles prepared by osmotic lysis are virtually devoid of soluble cytoplasmic proteins. Thus, all of the enzymatic activity associated with the vesicles is a property of the cytoplasmic membrane. Vesicles prepared by osmotic lysis are approximately 1 m in diameter, and if they are handled gently, each bacterial cell gives rise to one vesicle (i.e., they are analogous to red cell ghosts). However, mechanical stress during preparation may lead to pinching off of smaller sealed vesicles. Once frozen in liquid nitrogen, vesicles are stable for an indefinite period of time.

6. Figure 5. Freeze-fracture electron micrographs of membrane vesicles showing convex and concave surfaces. Left. Outer surface of membrane, fracture plane and view of inner leaflet of bilayer with membrane-embedded proteins appearing as bumps. Right. Outer leaflet of bilayer viewed from inside with pits where membrane proteins have been removed with the inner leaflet. Convex and concave surfaces always exhibit the same “texture”, and the convex surface is identical to the appearance of the cytoplasmic membrane in intact cells. Therefore, vesicles prepared by osmotic lysis retain same orientation as membrane in intact cell [i.e., they are right-side-out (RSO)]. This conclusion is supported quantitatively by binding studies with antibodies to proteins known to be on the cytoplasmic face of the membrane. By comparing intact and disrupted vesicles, no more than 2% of the vesicles prepared by osmotic lysis are either inverted or sufficiently leaky to allow antibody access to the cytoplasmic surface.
It is also possible to obtain vesicle preparations that are inside-out (ISO). However, ISO vesicles are about one-tenth the diameter of RSO vesicles.

7. Figure 6. Different types of active transport systems
Figure 6. Different types of active transport systems. Left, the phosphoenolpyruvate carbohydratephosphotransferase system, found in bacteria only, is a multi-component system that catalyzes vectorial phosphorylation of certain sugars. Also plays important role in regulation of metabolism (catabolite repression). Middle, lac permease, an example of ion-gradient driven active transport on which we will concentrate. Right, ATP Binding Cassette (ABC) systems in which ATP hydrolysis is the driving force for transport. May function as multi-component systems with a periplasmic binding protein or with components fused in different combinations. These bacterial transport ATPases are homologous to eucaryotic ABC transporters involved in cystic fibrosis (CFTR) and multi-drug resistance (MDR).

8. Figure 7. Assaying transport by filtration, the “quick and easy” method. A radioactively tagged transport substrate is added to RSO vesicles ( mL) in the presence or absence of an energy source (see below). At a given time, the vesicles are separated from the surrounding medium by rapid dilution and vacuum (VAC) filtration through filters with a pore diameter smaller than the diameter of the vesicles. The rate of filtration must be very rapid in order to trap the accumulated substrate. The method is reasonably quantitative for charged and neutral substrates (but accumulation of weak acids, in particular, is drastically underestimated because of passive permeability in the protonated state).

9. Figure 8. Determining intravesicular (or intracellular) space
Figure 8. Determining intravesicular (or intracellular) space. A concentrated suspension of vesicles or cells of a known protein concentration is mixed with 3H2O or [3H]urea which distribute equally between the internal and external spaces and [14C]sucrose which is impermeant. The vesicles or cells are then centrifuged, the supernatant is aspirated, the walls of the tube are dried, the pellet is resuspended and an aliquot is assayed for 3H and 14C in a scintillation counter. The difference between the 3H space (i.e. the internal plus external spaces) and the [14C]sucrose space (the external space) is the internal volume.
Knowing the protein concentration, the internal volume can be expressed in mL per mg protein. From this value, substrates taken up by the vesicles or cells can readily be converted to an internal concentration. Since the external concentration is known (the amount added minus the amount taken up), the concentration gradient can be calculated.

10. Figure 9. A typical transport assay carried out in the absence (l) or presence (m) of D-lactate (left) and the demonstration that D-lactate is converted stoichiometrically into pyruvate (right). The discovery that D-lactate is by far the most effective energy source for active transport of a wide variety of substrates in E. coli membrane vesicles took about 15 years, was totally fortuitous and broke open the field (Left; Control, no energy source). In addition to the fact that D-lactate is converted stoichiometrically to pyruvate (Right; Total, lactate + pyruvate), all of the accumulated substrate can be recovered from the vesicles in unmodified form which is consistent with the observations showing that addition of excess non-radioactive substrate (s) or inhibitors such as dinitrophenol (DNP; ∆) causes rapid release of accumulated radiolabeled lactose (Left). Oxygen and an intact membrane-embedded electron transfer chain are required. In order to observe accumulation of lactose or other galactosides, the parent cells must be induced for the synthesis of lactose permease from the lacY gene, the second structural gene in the lac operon. Other transport systems such as those for many amino acids are constitutive.

11. D-lactate is converted to pyruvate by a peripherally bound, flavin-linked D-lactate dehydrogenase. The bacterial plasma membrane, like the mitochondrial inner membrane, contains a respiratory chain, and anoxia or various inhibitors of electron transfer block both the conversion of D-lactate to pyruvate and active transport. Thus, oxidation of D-lactate to pyruvate by D-lactate dehydrogenase is coupled to a membrane-embedded respiratory chain, and as will be shown subsequently, electron transfer leads to the generation of a proton electrochemical gradient that is used to perform work in the form of active transport.

12. Figure 10. Other ways to energize active transport in addition to D-lactate oxidation. (a) Artificial electron donors [ascorbate/phenazine methosulfate (ASC/PMS); dithiothreitol/ubiquinol-1]; anaerobic electron transfer to nitrate or fumarate; (b) Internal ATP hydrolysis; (c) Artificially imposed membrane potentials (DY) or pH gradients (DpH).
Left. Vesicles loaded with KPi are diluted into NaPi, and valinomycin, a K+-specific ionophore (see Lecture II) is added to make the membrane specifically permeable to K+ (like the resting potential in a nerve cell). Since Na+ and Pi are impermeant, an outwardly directed K+ flux generates a DY (interior negative) that will drive accumulation of various transport substrates.
Right. Vesicles loaded with acetate are diluted into buffer containing an impermeant acid like gluconate. Since only the protonated (uncharged) form of acetate permeant, acetic acid leaves the vesicles, depleting the inside of protons and generating a DpH (interior alkaline).
pH Gradients can also be generated with the ionophores nigericin which has a higher affinity for K+ than Na+ or monensin which has a higher affinity for Na+ than K+. As opposed to valinomycin, these ionophores catalyze the 1:1 exchange of protons for either K+ or Na+ across the membrane. Thus, vesicles containing one of these ionophores and loaded with cholinePi are diluted into either KPi or NaPi. K+ or Na+ influx then drives efflux of protons out of the vesicles with generation of DpH (interior alkaline).
The reverse DY or DpH can aslo be generated by reversing the gradients.

13. Figure 11. Each membrane vesicle catalyzes active transport.
A. By using an analogue of lactate [2-hydroxy-3-butenoic acid (vinylglycolate; VG)] which is transported by a lactate transport protein and then oxidized inside of the vesicles to a reactive species (2-keto-3-butenoic acid), it is shown that the rate limiting step for covalently labeling vesicles is transport.

14. B. As with other substrates, ASC-PMS markedly stimulates uptake of VG
B. As with other substrates, ASC-PMS markedly stimulates uptake of VG. However, as opposed to normal substrates, virtually all of the radiolabeled VG taken up is covalently bound to the membrane and is not released by unlabeled VG or by boiling. Furthermore, the rate of covalent labeling in the presence of ASC-PMS is markedly inhibited by DNP, a protonophore that dissipates the driving force for transport.

15. C. By using [3H]substrate at a very high specific activity it can be shown directly by radioautography in the electron microscope that every vesicle is labeled. Micrograph shown at the left is from vesicles labeled with VG in the presence of ASC-PMS. The number of vesicles labeled non-specifically with [3H]acetic anhydride is not significantly different.

16. The preparation and characterization of bacterial membrane vesicles as a model system in which to study active transport led to the development of similar cytoplasmic membrane vesicle systems from eucaryotic cells, intestinal and kidney epithelia, as well as vesicles from intracellular organelles such as chromaffin granules, synaptosomes, etc. However, since these membranes do not contain a respiratory chain, artificially imposed ion gradients or ATP are used to drive active transport.