Ubiquitinylation and Ubiquitin-dependent Proteolysis in Vertebrate Photoreceptors (Rod Outer Segments)

doi:10.1074/jbc.271.24.14473

Ubiquitinylation and Ubiquitin-dependent Proteolysis in Vertebrate Photoreceptors (Rod Outer Segments)

EVIDENCE FOR UBIQUITINYLATION OF G_t AND RHODOPSIN*

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

^‡ To whom correspondence should be addressed:
Lab. for Nutrition and Vision Research, USDA-JMHNRCA, Tufts University, 711 Washington St., Boston, MA 02111.
Tel.: 617-556-3155; Fax: 617-556-3344; E-mail: Taylor_c1{at}HNRC.Tufts.edu

Abstract

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 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 (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.

Footnotes

↵* This work was supported by National Institutes of Health Grant 08566 and by United States Department of Agriculture-Agricultural Research Service Contract 53–3K06-0-1. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ROS

rod outer segment(s)

G_tαβγ (G_t)

transducin

G_tα

α-subunit of transducin

G_tβ

β-subunit of transducin

G_tγ

γ-subunit of transducin

UPP

ubiquitin-dependent proteolytic pathway

Ub

ubiquitin

E1

ubiquitin-activating enzyme

E2

ubiquitin carrier protein

E3

ubiquitin-protein isopeptide ligase

mAb

monoclonal antibody

Ubal

ubiquitin-aldehyde

RPE

retinal pigment epithelium(ial)

NEPHGE

non-equilibrium pH gel electrophoresis

PAGE

polyacrylamide gel electrophoresis

AMP-PNP

5′-adenylyl imidodiphosphate.
↵² R. Bohnsack and A. Haas, personal communication.
↵³ C. M. Pickart, personal communication.
↵⁴ M. S. Obin, J. Jahngen-Hodge, and A. Taylor, manuscript in preparation.
↵⁵ Hickey, L., and Riezman, H. (1996) Cell 84, 277–287
- Received November 3, 1995.
- Revision received February 27, 1996.