Why alanine mutagenesis

Finally, the use of alanine stretch cartridges allows progressive insertion, as shown in the example below. The outline of the method is as follows Fig. Site-directed mutagenesis is first used to introduce a Pst I restriction site in the sequence of the gene of interest. The IPCR mutagenesis method was chosen for its simplicity and high efficiency 2.

It should be noted that the Pst I and Pvu II sites of the vector bearing the cloned gene have to be eliminated beforehand, since the method relies on the use of these sites. In the substitution approach, this site is introduced so that the first codon of the five substituted ones is replaced by the GCT codon, and that the last one is replaced by GCA. The first base of the sixth codon is substituted by a guanine in order to create a complete Pst I site.

In the insertion approach, the GCTGCA sequence is introduced between two codons, and the first base of the second codon is changed to a guanine. Screening of the mutants bearing a single Pst I site is readily performed by restriction analysis. This cartridge comprises kanamycin resistance gene Kan R flanked on both sides by alanine codon sequences in which are embedded a Pvu II and a Pst I site Fig.

Transformants expressing kanamycin resistance are easily selected for. Extracted plasmids from these strains are then digested by Pvu II, ligated and transformed. The resulting clones are checked for there sensitivity to kanamycin and the plasmid DNA of one of these clones is sequenced. In all cases, the sequence showed the correct insertion of the alanine codons.

The design of the cartridge is such that its insertion in both orientations will yield the right insertion of the alanine codons. A second such cartridge has been created using the chloramphenicol resistance gene. This cartridge enables the insertion of four alanine codons in addition to the two created by the Pst I site instead of three for the kanamycin one. Any kind of other cartridges can thus be constructed, in order to insert the desired number of alanines through the use of a single Pst I site.

The insertion and substitution approaches have been tested on the TEM-1 b-lactamase. TEM-1 is one of the most commonly encountered plasmid-mediated b-lactamases, responsible for the hydrolysis of the b-lactam antibiotics, resulting in antibiotics resistance among many Gram-negative bacteria 3.

The 3D structure of this protein 4 shows a 86— loop between the h2 and h3 helix which seems to be out of the catalytic core of the enzyme, and so should not play any role in the catalytic pathway.

In order to check this hypothesis, a progressive alanine insertion has been realized in this loop. The Pst I site has first been created at the L position, substituting the leucine for an alanine, and inserting a second alanine.

This variant has been called TEM A. Four additional alanines have been introduced with the chloramphenicol cartridge, leading to the creation of the TEM —6A variant.

All the selected clones presented the correct sequences. Outline of the method, substitution approach. They were annealed in mM NaCl. The resulting vector was used as a source of the chloramphenicol resistance gene cartridge as a Pst I fragment.

The resulting vector was used as a source of the Pst I kanamycin resistance gene cartridge. Thus, inserting a two or six alanine stretch in the 86— loop of TEM-1 only has a minor impact on the enzymatic activity of the variants, confirming the minor role of this loop in the enzymatic process.

Ampicillin resistance of the various mutants as measured by antibiotic disc assay. In a second experiment, we substituted two turns of helix h5 of the TEM-1 enzyme by an alanine stretch. The 3D structure of TEM-1 hints that interactions between helix h5 and the main chain atoms of glutamic acid , a major catalytic residue, should play an important role in the enzymatic mechanism.

In accordance with our hypothesis, follow up experiments were performed and showed that the recombinant GST-fused C-terminal end of Gpm6a of 30 amino acids coimunoprecipitates with Coronin 1a using anti-coronin 1a antibody Supplementary Figure S4. Although additional future work is required to further characterize the interaction, these preliminary data prove the relevance of our findings.

Alternatively, it is possible that by mutating the residues we identified as functionally critical post-translational modifications or structural motifs that participate in the filopodium formation process are lost. Analysis of Gpm6a sequence revealed various signaling motifs that would be disturbed by alanine substitution of these residues. First, K and K are predicted as sites of ubiquitination.

In neurons, this pathway plays multiple roles and has been described as an emergent mechanism for regulating synapse function and plasticity Mabb and Ehlers, For example, ubiquitination of AMPA receptors regulates the intracellular sorting of receptors to late endosomes for degradation in lysosomes Widagdo et al. In addition, a variation of this consensus mono-leucine sorting motif EEXXXL was identified within the cytoplasmic domain of amphiregulin where it regulates biosynthetic delivery of amphiregulin to the basolateral surface Gephart et al.

The substitution of E or D with alanine in our study did not affect filopodium formation implying the notion that YEDI motif is dispensable for the process of filopodium formation. Accordingly, previous work by Formoso and coworkers showed that the replacement of the tyrosine residue at position by alanine affects only neurite extension but not filopodium formation Formoso et al.

On the other hand, the YA mutation was shown to totally abolish Gpm6a internalization induced by the monoclonal antibody without interfering in its immunodetection which led to the conclusion that Gpm6a endocytosis is mediated through the YEDI motif Garcia et al. Published works on sorting of plasma membrane proteins demostrated that there are proteins such as transferrin receptor TfR that use different sorting signals at trans Golgi network TGN and endosomes during biosynthetic delivery and post-endocytic recycling to the plasma membrane Odorizzi and Trowbridge, In this context, we can speculate that Gpm6a trafficking may use different motifs and different adaptors at each location in biosynthetic or recycling pathway.

On the other hand, in rat hippocampal neurons, overexpression of Gpm6a bearing simultaneous mutations of various putative intracellular phosphorylation sites including S did not affect formation of filopodia but did lower filopodium motility.

Nevertheless, the effect of mutation of individual intracellular phosphorylation sites was not addressed by this study Brocco et al. A monomeric protein named PACS-1 phosphofurin acidic cluster sorting protein 1 was identified that binds to acidic clusters in a CK2 phosphorylation dependent manner and functions as a connector that links the phosphorylated acidic clusters to the clathrin-dependent sorting machinery Wan et al.

Taken together, using alanine scanning mutagenesis we identified amino acids in the C-terminal cytosolic end of Gpm6a essential in the process of filopodium formation that are predicted as parts of sorting motifs. In this context, diminished surface expression of mutant proteins we observe in our study could indicate that Gpm6a trafficking is disturbed by replacement of these residues.

On the other hand, decreased total protein expression of mutant proteins KA and EA suggests their degradation probably due to the destabilizing effect on protein folding. Future work is required to establish whether and which biosynthetic or internalization pathways are affected by individual mutations. Membrane traffic systems in non-neuronal cells contribute to cell morphogenesis and appear to be driving factors for cell polarization.

In neurons, filopodial processes were identified as one of the hot spots of active membrane remodeling where endocytic membrane retrieval initiates in the growth cone during axon extension Hines et al. Moreover, membrane trafficking from recycling endosomes is required for the growth and maintenance of spines and regulated membrane trafficking of postsynaptic neurotransmitter receptors has emerged as a central mechanism for synapse development and modification Ehlers, ; Park et al.

Consistently, Gpm6a has been suggested to facilitate micro-opioid receptor and a number of other GPCRs endocytosis and recycling Wu et al. Our observation that deletion of the C-terminal, but not the N-terminal, cytosolic domain diminishes colocalization of Gpm6a with clathrin further points to the functional significance of the C-terminal end of Gpm6a and to the involvement of clathrin mediated trafficking events in the process of filopodium formation induced by Gpm6a.

BF conceived and designed the study. BF wrote the paper. NR wrote sections of the manuscript. All authors contributed to manuscript revision, read and approved the submitted version. The funding sources had no role in study design, acquisition, and interpretation of data or writing of the report. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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