Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments) - Evidence for ubiquitinylation of G(t) and rhodopsin

In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pi 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (G(t)) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and G, were readily solubilized in buffer lacking GTP, These data are consistent with ubiquitinylation of G(t) and corroborate previous cell-free experiments identifying G(t) as a substrate for ubiquitin-dependent proteolysis (Obin, M. S,, Nowell, T,, and Taylor, A. (1994) Biochem. Biophys. Res, Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to G(t), included (i) the presence in ROS membranes ''stripped'' of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of I-125-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a ''ladder'' of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these I-125-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of I-125-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (El); (ii) four ubiquitin carrier proteins (E2(14K), E2(20K), E2(25K), and E2(35K)) and pronounced activity of E2(14K), an enzyme required for ''N-end rule'' proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass I-125-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties, Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.