Figure 3
From: Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein
Neu3 is S-acylated. (a) A schematic diagram of the acyl-biotin exchange (ABE) assay. (1) Irreversible blockade of free thiol groups using N-ethylmaliemide (NEM); (2) Specific cleavage of thioester bonds and unmasking of acylated cysteines by hydroxylamine (HAM). In this step, omission of HAM served as a negative control; (3) Labeling of the S-acylated cysteines using the thiol-reactive biotinylation reagent HPDP-biotin; (4) Pull-down of the biotinylated proteins with streptavidin agarose beads (SA). (b) Cell lysates from NEU3-transfected cells were subjected to ABE analysis. For the negative control, HAM was substituted by Tris buffer (-HAM). Cav-1 was used as a marker of a known S-acylated protein. Neu3 purified from HEK-293 cells was also subjected to ABE analysis. (c) Effect of HAM deacylation of Neu3 on its membrane association. Membrane fractions (P, second lane) were treated with Tris buffer (control) or with HAM. After treatment, membranes were collected by ultracentrifugation, and membrane-bound (P, fourth and sixth lanes) and solubilized (S, third and fifth lanes) proteins were analyzed by Western blotting using anti-Neu3 antibody. As the two covalently-bound fatty acids of GAP43 are directly involved in its membrane association, GAP43 was used as a marker of a protein released into the supernatant after treatment with HAM. Cav-1 was used as a marker of an S-acylated protein not released after HAM treatment.
