Membrane‐binding sites for acyl carrier protein in <i>Escherichia coli</i>

Bayan, Nicolas; Therisod, Hélène

doi:10.1016/0014-5793(89)80963-9

Cited by 5 publications

(3 citation statements)

References 18 publications

(21 reference statements)

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“…The proportion of S. erythraea ACP released into the supernatant was approximately equal to the proportion of E. coli ACP released. These results do not necessarily support the proposal (Van der Bosch et al, 1970;Bayan and Therisod, 1989) that ACP is associated with the cytoplasmic membrane, but they do indicate that the freeze-thaw method of releasing ACPs from E. coli cells may be of general applicability. In the first instance, the induced E. coli K38 (pGPI-2, pFEX-1) cells were thawed into buffer A that contained no DTT.…”

Section: Resultscontrasting

confidence: 67%

“…both thioredoxin and elongation factor Tu are associated with the inner surface of the cytoplasmic membrane Lunn and Pigiet, 1982). Since evidence has been reported that E. coli ACP is associated with the cytoplasmic membrane (Van der Bosch et al, 1970;Bayan and Therisod, 1989), it was thought that it too might be selectively released from E. coli by osmotic shock. Attempts to purify the S. erythraea ACP by osmotic-shock release into various concentrations of sucrose solutions (15-40 %, w/v) were unsuccessful.…”

Section: Resultsmentioning

confidence: 99%

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Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

Morris

Revill

Staunton

et al. 1993

Biochemical Journal

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Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

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Section: Resultscontrasting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

Morris

Revill

Staunton

et al. 1993

Biochemical Journal

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“…Specifically, osmotically shocked cells have been shown to retain a small (8.5-kDa) soluble acyl carrier protein (9), thus seemingly contradicting our conclusion. However, this protein might be prevented from exiting cells by binding the plasma membrane (2,3). The other apparent contradiction is presented by a large EntF polypeptide (142 kDa) that was shown to be partially released upon osmotic shock (9).…”

Section: Discussionmentioning

confidence: 99%

Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

Vázquez‐Laslop

Lee

et al. 2001

J Bacteriol

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Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises ϳ10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells.The procedure of osmotic shock was originally introduced for the purpose of extracting periplasmic enzymes of Escherichia coli (20). In this procedure, bacterial cells are preincubated in a hyperosmotic sucrose solution supplemented with EDTA to permeabilize their outer membrane and then transferred into a solution of low osmolarity. The resulting osmotic pressure within shocked cells was believed to cause extrusion of the contents of the periplasm. Later experiments have shown, however, that osmotically shocked bacteria, while remaining viable, also release a number of cytoplasmic molecules, including ions, metabolites (22), and certain proteins. In fact, the major protein released from the shocked cells is a cytoplasmic constituent, translation elongation factor EF-Tu, at least half of which is extracted by the osmotic shock procedure (12, 13). The subset of released cytoplasmic proteins also includes thioredoxin (1, 17), molecular chaperone DnaK (6,8), and proteins involved in the biosynthesis of enterobactin (9), among others (5). Some foreign proteins expressed in the cytoplasm of E. coli have also been successfully extracted by the osmotic shock procedure (16, 23).It has been unclear what determines the selectivity of protein release from osmotically shocked bacteria, since the released cytoplasmic proteins share no apparent common characteristics distinguishing them from the majority of proteins, which are retained by the cells. How these proteins leave the cytoplasm in the absence of cell lysis has also remained an enigma. The most frequently entertained hypothesis postulates that these proteins normally concentrate in a hypothetical "osmotically sensitive compartment" of the bacterial cytoplasm, which is presumably adjacent to the zones of adhesion between the plasma membrane and the outer membrane and, therefore, is selectively extruded from the shocked cells (4, 8-10, 18, 23).This hypothesis leaves open, however, a s...

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A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli

Gully¹,

Bouveret²

2006

Proteomics

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In prokaryotes, acyl carrier protein (ACP) is a cofactor central to a myriad of syntheses, including fatty acid and phospholipid synthesis. To fulfill its function, ACP must therefore interact with a multitude of different enzymes, which includes the thioesterase YbgC. We found a specific interaction between ACP and YbgC whose thioesterase activity has been demonstrated in vitro on acyl-CoA derivatives, but whose physiological function in bacteria remains unknown. Therefore, YbgC could be a thioesterase active on some specific acyl-ACPs. We then assigned a function to the ACP/YbgC pair by employing a proteomic approach derived from tandem affinity purification, the split tag method. This technique allowed us to purify proteins interacting with ACP and YbgC proteins at the same time. Interactions with PlsB, a sn-glycerol-3-phosphate acyltransferase and PssA, a phosphatidylserine synthase, were identified and validated, showing that YbgC is involved in phospholipid metabolism. Furthermore, using an in vivo bacterial two-hybrid interaction analysis, we showed for the first time that enzymes of the phospholipid synthesis pathway form a complex in the inner membrane. Taken together, these results describe an integrated protein network that could be involved in the coordination of phospholipid metabolism.

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Membrane‐binding sites for acyl carrier protein in Escherichia coli

Cited by 5 publications

References 18 publications

Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli

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