Thyroxine dynamically regulates levels of type II iodothyronine 5-deiodinase (5D-II) by modulating enzyme inactivation and targeting the enzyme to different pathways of internalization. 5D-II is an ϳ200-kDa multimeric protein containing a 29-kDa substrate-binding subunit (p29) and an unknown number of other subunits. In the absence of thyroxine (T 4 ), p29 is slowly endocytosed and transported to the lysosomes. T 4 treatment rapidly activates an actin-mediated endocytotic pathway and targets

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... enzyme to the endosomes. In this study, we have characterized the influence of T 4 on the intracellular trafficking of 5D-II. We show that T 4 accelerates the rate of 5D-II inactivation by translocating the enzyme to the interior of the cell and by sequestering p29 in the endosomal pool without accelerating the rate of degradation of p29. This dichotomy between the rapid inactivation of catalytic activity and the much slower degradation of p29 is consistent with the reuse of p29 in the production of 5D-II activity. Immunocytochemical analysis with a specific anti-p29 IgG shows that pulse affinity-labeled p29 reappears on the plasma membrane ϳ2 h after enzyme internalization in the presence of T 4 , indicating that p29 is recycled. Despite the ability of p29 to be recycled in the T 4 -treated cell, 5D-II catalytic activity requires ongoing protein synthesis, presumably of another enzyme component(s) or an accessory enzyme-related protein. In the absence of T 4 , enzyme inactivation and p29 degradation are temporally linked, and pulse affinity-labeled p29 is internalized and sequestered in discrete intracellular pools. These data suggest that T 4 regulates fundamental processes involved with the turnover of integral membrane proteins and participates in regulating the inter-relationships between the degradation, recycling, and synthetic pathways. . 1 The abbreviations used are: 5ЈD-II, type II iodothyronine 5Ј-deiodinase; T 4 , thyroxine; T 3 , 3,5,3Ј-triiodothyronine; rT 3 (reverse T 3 ), 3,3Ј,5Јtriiodothyronine BrAcT 4 , N-bromoacetyl-L-thyroxine; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; BFA, brefeldin A; Bt 2 cAMP, dibutyryl cAMP.