Abstract
Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two "complementary" forms of rat AANAT are targeted for degradation by two "complementary" branches of the N-end rule pathway. Specifically, the N(α)-terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt-acetylated N-terminal Met residue by the Ac/N-end rule pathway, whereas the non-Nt-acetylated AANAT is targeted by the Arg/N-end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine-to-arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer-lived than its rat counterpart and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N-terminal sequences.
Highlights
For example, the Met residue of the Nterminal Met-Leu-Ser sequence of wild-type rat AANAT (Fig. 1C) is retained in mature MLSrAANAT, inasmuch as Leu is larger than Val
Given the previously demonstrated usefulness of employing genetic tractability of S. cerevisiae for understanding the targeting of mammalian N-end rule substrates [60, 66], we carried out the present study by expressing AANAT test proteins in human HEK293T cells and in S. cerevisiae
We found that the degradation of the wild-type rat MLSrAANAT3f (t1⁄2 Ͻ 5 min in wild-type S. cerevisiae) was not significantly impaired in doa10⌬ cells, in comparison with wild-type cells (Fig. 3, D and F)
Summary
Wild-type Rat and Human AANATs and Their Mutants— The start (AUG) codon-encoded N-terminal Met residue of nascent proteins is cotranslationally cleaved off by ribosomeassociated Met-aminopeptidases (MetAPs) if a residue at position 2, to be made N-terminal by the cleavage, is not larger than Val [41, 74]. Because the N-terminal Met residue in the Met-Leu-Ser sequence of the wild-type rat MLSrAANAT3f would be likely to be Nt-acetylated in vivo [59, 62, 63], the rapid degradation of MLSrAANAT3f (t1⁄2 Ͻ 5 min) (Fig. 2, B and D) suggested the involvement of the Ac/N-end rule pathway (Fig. 1A) To address this possibility, we constructed the non-Nt-acetylatable (M)PLSrAANAT3f mutant (see item iv above). The latter result suggested that (residual) polyubiquitin chains observed in cells that expressed MLSrAANAT3Khzaero may be of a kind (e.g. see Ref. 85) that do not contribute to the proteasome-dependent degradation of MLSrAANAT3Khzaero In agreement with this interpretation, the lysine-lacking MLSrAANAT3Khzaero mutant was expressed at significantly higher steady-state levels than the wild-type rat MLSrAANAT3ha in the absence of MG132 (in DMSO-treated cells) (Fig. 6E, bottom), suggesting that the absence of Lys residues stabilized MLSrAANAT3Khzaero against degradation throughout life histories of MLSrAANAT3Khzaero molecules, including, possibly, their degradation during or immediately after their synthesis.
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