Posttranslational translocation

The rate of maturation of pre - b - lactamase was determined by measuring the decrease in the amount of pre- 8 lactamase after one minute labeling interval. There was no significant difference between the control and lincomycin treated cultures, indicating that posttranslational translocation is not involved in the stimulation. 

Translocation is believed to occur posttranslation-ally, after the matrix proteins are released from the cytosolic polyribosomes. Interactions with a number of cytosolic proteins that act as chaperones (see below) and as targeting factors occur prior to translocation. 

In the ER translocation system, most of mammalian proteins are likely to use the SRP-dependent pathway, whereas in yeast the SRP-independent pathway as well as the SRP-dependent pathway are heavily used. The SecB-dependent pathway in bacteria seems to correspond with this SRP-independent pathway, which is posttranslational. Instead of SecB, various proteins including BiP, Sec62p, and Sec63p are involved. 

The majority of cases of cystic fibrosis result from deletion of phenylalanine at position 508 (AF508), which interferes with proper protein folding and the posttranslational processing of oligosaccharide side chains. The abnormal chloride channel protein (CFTR) is degraded by the cytosolic proteasome complex rather than being translocated to the cell membrane. Other functional defects in CFTR protein that teaches the cell membrane may also contribute to the pathogenesis of cystic fibrosis. 

To function, Ras must be attached to the plasma membrane. Translocation from the cytoplasm to membrane requires a series of posttranslational modifications that begin with farnesylation of the cysteine residue, the fourth amino acid residue from the C terminus of the protein, by famesyl protein transferase (FPTase) (64). Attachment of the hydrophobic 15-carbon lipid farnesyl group allows Ras molecule insertion into the plasma membrane and is crucial for Ras signaling activity and transformation properties. As farnesylation is required for oncogenic Ras function, FPTase inhibitors (FTIs) are obvious candidate antineoplastic agents. Several drugs that inhibit Ras farnesylation are at various stages of clinical development (65). 

The enzymatic specificity of diphtheria toxin deserves special comment. The toxin ADP-ribosylates EF-2 in all eukaryotic cells in vitro whether or not they are sensitive to the toxin in vivo, but it does not modify any other protein, including the bacterial counterpart of EF-2. This narrow enzymatic specificity has called attention to an unusual posttranslational derivative of histidine, diphthamide, that occurs in EF-2 at the site of ADP-ribosylation (see fig. 1). Although the unique occurrence of diphthamide in EF-2 explains the specificity of the toxin, it raises questions about the functional significance of this modification in translocation. Interestingly, some mutants of eukaryotic cells selected for toxin resistance lack one of several enzymes necessary for the posttranslational synthesis of diphthamide in EF-2 that is necessary for toxin recognition, but these cells seem perfectly competent in protein synthesis. Thus, the raison d etre of diphthamide, as well as the biological origin of the toxin that modifies it, remains a mystery. 

Fig. 10. Cell-free synthesis of t-PA glycoforms. The niRNA coding for t-PA was translated in a rabbit reticulocyte lysate in the presence of dog pancreas microsomes. Microsonies were isolated posttranslationally and the translocated, glycosylated products were separated by SDS-PAGE. Translation was carried out under conditions that either prevented (lane 2) or allowed (lane 3) proper folding of the t-PA molecule, yielding enzymatically active protein that was sensitive to natural inhibitors and stimulators.

Figure 29.35. Blocking of Translocation by Diphtheria Toxin. Diphtheria toxin blocks protein synthesis in eukaryotes by catalyzing the transfer of an ADP-ribose unit from NAD+ to diphthamide, a modified amino acid residue in elongation factor 2 (translocase). Diphthamide is formed by a posttranslational modification (blue) of a histidine residue.

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