Membrane-targeting Domain Research Articles

Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis

Neuromodulin (GAP-43) is a neurospecific calmodulin binding protein which is implicated in neuronal growth and regeneration. It is concentrated in neuronal growth cones and associates with membranes through the palmitoylation of the N-terminal peptide MLCCMRRTK at Cys-3 and Cys-4. In the present study, we have identified critical amino acid residues required for palmitoylation and membrane association of neuromodulin in vivo. Several neuromodulin constructs with point mutations were tested for membrane association and palmitoylation. Wild-type neuromodulin expressed in COS-7 cells incorporated [3H]palmitic acid, whereas a mutant in which both Cys-3 and Cys-4 were substituted with glycine was not palmitoylated in vivo. Mutant proteins in which either Cys-3 or Cys-4 was substituted with leucine incorporated 75% and 25% of [3H]palmitic acid, respectively, compared to wild-type neuromodulin. The relative distribution of mutant neuromodulins expressed in COS-7 cells was quantitated by immunoblot analysis of the membrane and cytosolic fractions. There was a general correlation between membrane association of mutant neuromodulins and the extent to which they were palmitoylated in vivo. Additional point mutations in the acylation domain of neuromodulin indicated that a short hydrophobic amino acid sequence N-terminal to Cys-4 may be required for optimal palmitoylation and membrane association. We conclude that Cys-4 is critical for the palmitoylation and membrane association of neuromodulin.

Insertion and folding of the amino-terminal amphiphilic signal sequences of the mannitol and glucitol permeases of Escherichia coli.

Peptides which correspond to the NH2-terminal 23 or 22 residues of the mannitol and glucitol permeases (enzymes IImtl and IIgut of the bacterial phosphotransferase system; mtl-23 and gut-22) and which are believed to function in envelope targeting were synthesized chemically, and their interactions with lipid model membranes were studied. Both wild-type peptides penetrated phospholipid monolayers up to high surface pressures, and partition constants of 8.0 x 10(4) M-1 and 4.2 x 10(4) M-1, respectively, were derived from the incorporation isotherms of mtl-23 and gut-22 with monolayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine at 32 mN/m or bilayers of the same lipid. The mtl-23 peptide was highly alpha-helical in trifluoroethanol, sodium dodecyl sulfate, lysolecithin, or vesicles of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylglycerol, with estimated percentages of alpha-helix ranging between 60 and 85%. The interactions with model membranes of several single site mutants (S3P, D4P, and D4K) of mtl-23 which were defective in properly assembling the mannitol permease in the cytoplasmic membrane of Escherichia coli were also studied. The contents of alpha-helix of these peptides in detergent micelles or phospholipid bilayers were not significantly changed compared with those of the wild type, suggesting that the amphiphilic NH2-terminal membrane-targeting domain could still be formed in these mutants. However, the mutants which contained a proline in positions 3 or 4, i.e. NH2-terminal to the proposed amphiphilic alpha-helix, partitioned into phospholipid monolayers with partition constants that were 2 or 4 times smaller than those of the wild type. Based on these data, a model of the amphiphilic structure of the NH2-terminal domain of the mannitol permease is discussed. This domain may interact physiologically with amphiphilic interfaces of lipids and/or proteins during membrane insertion.

Cloning and Characterization of the Rat Gene Encoding GAP‐43

GAP-43 is a gene expressed only in the nervous system. The protein product is believed to be important to neuronal growth and plasticity. Most, and likely all, neurons express high levels of GAP-43 during periods of neurite elongation. To initiate studies of GAP-43 gene regulation we have cloned the rat gene encoding GAP-43. The GAP-43 gene includes three exons. The first exon encodes only the amino terminal 10 amino acids, which corresponds to the membrane targeting domain of GAP-43. The second exon encodes a putative calmodulin binding domain and a protein kinase C phosphorylation site. The 5'-flanking sequence is unusual in that it lacks CAAT or TATA elements, and directs RNA transcription initiation from several sites. Some of the transcription start sites are used to a different degree in the central and peripheral nervous systems.