REVERSAL OF SIGNAL-MEDIATED CELLULAR RETENTION BY SUBUNIT ASSEMBLY OFHUMAN ACETYLCHOLINESTERASE

The interrelationship between signal-mediated endoplasmic reticulum re tention and control of subunit assembly in secreted complex proteins w as examined in recombinant 293 cells expressing human acetylcholineste rase (HuAChE). This was achieved by analyzing the mutual effects of co -residing retention and dimerization signals on enzyme secretion by tr ansfected cells. The function of putative signals within the COOH-term inal tetrapeptide CSDL of HuAChE was examined by site-directed mutagen esis. The CSDL tetrapeptide carries the free cysteine (Cys-580) involv ed in subunit assembly yet it fails to function as a KDEL-type retenti on signal. This was demonstrated by mutations that increase similarity to the canonical retention signal (substitution of CSDL by KSDL) or t hose that deviate from it (substitution to CSAL). Cells expressing bot h types of mutants exhibited cell-associated HuAChE levels identical t o that of wild type enzyme. Appendage of an engineered KDEL retention signal to a dimerization-impaired HuAChE subunit (the C580A mutant) re sulted in intracellular retention of large amounts of fully active enz yme not prone to proteolytic degradation. On the other hand, attachmen t of KDEL to a native, dimerization-competent HuAChE polypeptide did n ot lead to intracellular retention and allowed efficient secretion of enzyme to the cell growth medium. Yet, appendage of KDEL to the native HuAChE led to some retardation in the transport of enzyme molecules t hrough the Golgi apparatus, as manifested by increase in cellular popu lation of endo Ii-resistant dimers, when compared with wild type enzym e. Taken together, these results indicate (a) that subunit dimerizatio n mediated by the COOH-terminal cysteine of HuAChE can reverse the sig nal-mediated retention by masking recognition of KDEL by its cognate r eceptor and (b) that the native sequences of the acetylcholinesterase subunit polypeptide do not appear to function as a coupled retention/d imerizatian signal in the control of secretion of assembled enzyme mol ecules.