Study of domains and function specificity of rab3a by construction of chimeric proteins

ROGGERO, CARLOS MARCELO; LOPEZ, CECILIA; MESA, ROSANA; YUNES, ROBERTO; MAYORGA, LUIS S.

Villa Carlos Paz, Cordoba

Congreso; XXXVIII Reunión Nacional Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).; 2002

Small GTPases of the Rab family are involved in specific stages of the vesicular traffic. Rabs work like molecular switches that alternate between an active, GTP-bound form, and an inactive, GDPbound form. Rab3 proteins are present in exocytotic granules, Rab22A is associated with early and late endosomes, whereas RabSA is restricted to early endosomes. To establish the domains of Rab3A that confer specificity of function we designed chimeric proteins combining the C- and N- terminus domains of Rab3A, RabSA, and Rab22A. Rab3A is present in human spermatozoa and stimulates the acrosome reaction in streptolysin 0-permeabilized cells. The spermatozoa do not have the capacity to endocyte and therefore lack the relevant effector proteins necessary for Rab22A and RabSA activity. cDNAs of the Rab proteins were engineered in plasmids, expressed as GST-fusion proteins, and their function tested on the acrosome reaction. Chimeras RabS 1-7838 1-22 1 and Rab22 1-6338 1-22 1 which bear Rab3 C-terminus, stimulated the acrosome reaction to an extent similar to full length Rab3A. Contrastingly, chimeras Rab3 1-80579-2 15 and Rab3 1-802264- 195 inhibited calcium-triggered acrosome reaction. The N-terminus would inhibit exocytosis by engaging effectors -and therefore competing with the endogenous protein- in fusion-incompetent complexes. The C-terminus portion of Rab3A is responsible for its exocytosis- triggering activity, perhaps by activating effectors that tether the acrosome to the plasma membrane.