Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt 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 (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt 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 Gt, 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 125I-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 (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, 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.
Obin
M S
MS
Laboratory for Nutrition and Vision Research, United States Department of Agriculture-Jean Mayer Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111, USA.
Jahngen-Hodge
J
J
Nowell
T
T
Taylor
A
A
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism / Q000528:radiation effects; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D007700:Kinetics; D008025:Ligases; D008027:Light; D009097:Multienzyme Complexes / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012243:Rhodopsin / Q000302:isolation & purification / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013869:Thiolester Hydrolases / Q000378:metabolism; D015293:Transducin / Q000302:isolation & purification / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism*; D014714:Vertebrates
D002352:Carrier Proteins; D007457:Iodine Radioisotopes; D009097:Multienzyme Complexes; D014452:Ubiquitins; C046288:ubiquitin carrier proteins; D012243:Rhodopsin; D044763:Ubiquitin-Conjugating Enzymes; D013869:Thiolester Hydrolases; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D015293:Transducin; D008025:Ligases