Abstract
The cold osmotic shock procedure releases a protein (GLPT) from the cell envelope of Escherichia coli that is related to the transport of sn‐glycerol‐3‐phosphate in this organism. The evidence for this correlation is as follows: (1) GLPT is under the regulatory control of the glpR gene. (2) Some glpT mutants that were isolated as phosphonomycin resistant clones do not synthesize GLPT. Revertants of these mutants (growth on sn‐glycerol 3‐phosphate) again synthesize GLPT. (3) Some amber mutations in glpT reduce the amount of GLPT while suppressed strains produce normal amounts. (4) Transfer of a plasmid carrying the glpT genes into a strain lacking GLPT and sn‐glycerol‐3‐phosphate transport restores both functions in the recipient. Transport and GLPT synthesis in the plasmid carrying strain are increased 2‐ to 3‐fold over a fully induced wild‐type strain, but appear to be constitutive. GLPT is a soluble protein of molecular weight 160,000 composed of 4 identical subunits. The 160,000 molecular weight complex is stable in 1% sodium dodecylsulfate at room temperature. Upon boiling in 1% sodium dodecylsulfate GLPT dissociates into its subunits. Likewise, 8 M urea at room temperature dissociates GLPT into its subunits. Dialysis of dissociated GLPT against phosphate or Tris‐HCl buffer, pH 7.0, allows renaturation to the tetrameric form. The protein is acidic in nature (isoelectric point 4.4).
In contrast to the typical transport‐related periplasmic‐binding proteins, no conditions could be found where pure GLPT exhibited binding activity toward its supposed substrate, sn‐glycerol‐3‐phosphate.
In vivo new appearance of transport activity for sn‐glycerol‐3‐phosphate transport occurs only shortly before cell division. However, GLPT synthesis does not fluctuate during the cell cycle. The available evidence indicates a cell‐division‐dependent processing of GLPT in the cell envelope as a reason for the alteration in transport activity.
Transport in whole cells is sensitive to the cold osmotic shock procedure, demonstrating the participation of an essential periplasmic component. However, isolated membrane vesicles that are devoid of periplasmic components, including GLPT, are fully active in sn‐glycerol‐3‐phosphate transport. Therefore, we conclude that GLPT is essential in overcoming a diffusion barrier for sn‐glycerol‐3‐phosphate established by the outer membrane. Attempts to isolate mutants that are transport negative in whole cells due to a defect in GLPT but are active in isolated membrane vesicles have failed so far. All GLPT mutants tested, whether or not they synthesize GLPT, are not active in isolated membrane vesicles.
Iodination of whole cells with [^125^I] followed by osmotic shock reveals that several shock‐releasable proteins including GLPT become radioactively labeled. This indicates that some portions of GLPT are accessible to the external medium.