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Volume 271, Number 24, Issue of June 14, 1996 pp. 14473-14484
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Ubiquitinylation and Ubiquitin-dependent Proteolysis in Vertebrate Photoreceptors (Rod Outer Segments)
EVIDENCE FOR UBIQUITINYLATION OF G_t AND RHODOPSIN

(Received for publication, November 3, 1995, and in revised form, February 27, 1996)

From the Laboratory for Nutrition and Vision Research, United States Department of Agriculture-Jean Mayer Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111

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_t 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 <sup>125</sup>I-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 ¹²⁵I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of ¹²⁵I-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 (E1); (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 ¹²⁵I-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.

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