Journal: The Journal of Biological Chemistry
Article Title: Evolutionary duplication of the leishmanial adaptor protein α-SNAP plays a role in its pathogenicity
doi: 10.1016/j.jbc.2025.108427
Figure Lengend Snippet: Functional complementation of L. donovani putative α-SNAPs in yeast sec17–1 mutant strain and the critical role of an N-terminally located phosphorylatable-serine/glutamate in the functionality of α-SNAP . A , both leishmanial α-SNAPs, α-SNAP1, and α-SNAP2, were tested for their abilities to functionally substitute yeast α-SNAP Sec17 within temperature-sensitive sec17-1 mutant yeast strain RSY269. The mutant yeast cells were transformed with the constructs p426GPD- Ld α-SNAP1, p426GPD- Ld α-SNAP2, p426GPD- SEC17 (positive control), and empty p426GPD vector (negative control). The transformants were spotted on yeast synthetic medium lacking uracil (Ura d/o) plates. Subsequently, their growth was monitored at permissive (30 °C) and nonpermissive temperatures (37 °C) for 2 days. B , multiple sequence alignment of N-terminal 15 amino acids of Leishmania , yeast, and Toxoplasma α-SNAP orthologs to search for the well-defined phosphorylatable-serine residue in α-SNAP paralogs of trypanosomatid parasite Leishmania donovani . The black box and asterisk indicate conserved phosphorylatable-serine/glutamate residue at the sixth position of proteins. The red box represents the substitution of conserved serine/glutamate with aspartate at the sixth position of Ld α-SNAP2. The yellow box shows alanine-7 of Ld α-SNAP2 aligned with conserved serine-6/glutamate-6 in multiple sequence alignment. Cross-species complementation in yeast hypomorph using the phosphomimetic, phosphorylatable, non-phosphorylatable, and similarly charged residue substituted α-SNAP mutants. Constructs containing: C , the wild-type α-SNAP1, phosphomimetic: α-SNAP1 (S6D) , α-SNAP1 (S6E) and non-phosphorylatable: α-SNAP1 (S6A) substitution mutants, D , the wild-type Sec17 and its mutants: Glutamate 6 Serine, Glutamate 6 Aspartate and Glutamate 6 Alanine, E , the wild-type α-SNAP2, phosphorylatable α-SNAP2 (D6S) , and a very similar negatively charge substituted i.e. α-SNAP1 (D6E) mutant, and. F , phosphorylatable α-SNAP2 (A7S) mutants, were individually transformed in temperature-sensitive yeast strain RSY269. Growth of these transformants at both permissive (30 °C) and nonpermissive temperature (37 °C) was assessed by spotting serial dilutions on yeast synthetic medium lacking uracil (Ura d/o).
Article Snippet: The chromosomal sequences of α-SNAP genes from both wild-type yeast (MATα) and Leishmania (AG83) were first PCR-amplified using the primers listed in and then individually cloned under the control of a strong constitutive promoter, glyceraldehyde 3-phosphate dehydrogenase (GPD), into a 2μ plasmid, p426GPD (ATCC 87361), having URA3 as the yeast selectable marker gene.
Techniques: Functional Assay, Mutagenesis, Transformation Assay, Construct, Positive Control, Plasmid Preparation, Negative Control, Sequencing, Residue
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