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The Cyclophilin-like Domain Mediates the Association of Ran-Binding Protein 2 with Subunits of the 19 S Regulatory Complex of the Proteasome

1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês

10.1074/jbc.273.38.24676

ISSN

1083-351X

Autores

Paulo A. Ferreira, Yunfei Cai, Diana Schick, Ronald Roepman,

Tópico(s)

Endoplasmic Reticulum Stress and Disease

Resumo

The combination of the Ran-binding domain 4 and cyclophilin domains of Ran-binding protein 2 selectively associate with a subset of G protein-coupled receptors, red/green opsins, uponcis-trans prolyl isomerase-dependent and direct modification of opsin followed by association of the modified opsin isoform to Ran-binding domain 4. This effect enhances in vivo the production of functional receptor and generates an opsin isoform with no propensity to self-aggregate in vitro. We now show that another domain of Ran-binding protein 2, cyclophilin-like domain, specifically associates with the 112-kDa subunit, P112, and other subunits of the 19 S regulatory complex of the 26 S proteasome in the neuroretina. This association possibly mediates Ran-binding protein 2 limited proteolysis into a smaller and stable isoform. Also, the interaction of Ran-binding protein 2 with P112 regulatory subunit of the 26 S proteasome involves still another protein, a putative kinesin-like protein. Our results indicate that Ran-binding protein 2 is a key component of a macro-assembly complex selectively linking protein biogenesis with the proteasome pathway and, thus, with potential implications for the presentation of misfolded and ubiquitin-like modified proteins to this proteolytic machinery. The combination of the Ran-binding domain 4 and cyclophilin domains of Ran-binding protein 2 selectively associate with a subset of G protein-coupled receptors, red/green opsins, uponcis-trans prolyl isomerase-dependent and direct modification of opsin followed by association of the modified opsin isoform to Ran-binding domain 4. This effect enhances in vivo the production of functional receptor and generates an opsin isoform with no propensity to self-aggregate in vitro. We now show that another domain of Ran-binding protein 2, cyclophilin-like domain, specifically associates with the 112-kDa subunit, P112, and other subunits of the 19 S regulatory complex of the 26 S proteasome in the neuroretina. This association possibly mediates Ran-binding protein 2 limited proteolysis into a smaller and stable isoform. Also, the interaction of Ran-binding protein 2 with P112 regulatory subunit of the 26 S proteasome involves still another protein, a putative kinesin-like protein. Our results indicate that Ran-binding protein 2 is a key component of a macro-assembly complex selectively linking protein biogenesis with the proteasome pathway and, thus, with potential implications for the presentation of misfolded and ubiquitin-like modified proteins to this proteolytic machinery. Ran-binding domain 4 cyclophilin cyclophilin-like domain glutathione S-transferase polyacrylamide gel electrophoresis polyvinylidene difluoride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid peptidyl prolyl cis-trans isomerase adenosine 5′-O-(thiotriphosphate) guanosine 5′-O-(thiotriphosphate) guanyl-5′-yl thiophosphate. G protein-coupled receptors or so called seven-transmembrane receptors constitute the largest gene family known to date (1Savarese T. Fraser C. Biochem. J. 1992; 283: 1-19Crossref PubMed Scopus (441) Google Scholar, 2Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar, 3Bargmann C.I. Cell. 1997; 90: 585-587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). It is estimated that this family consists of 5,000 members (4Intelligent Drug Design (1996) Nature 384, (suppl.) 5Google Scholar). They play a key role in mediating environmental ques to the intracellular signaling machinery (1Savarese T. Fraser C. Biochem. J. 1992; 283: 1-19Crossref PubMed Scopus (441) Google Scholar, 2Gudermann T. Nurnberg B. Schultz G. J. Mol. Med. 1995; 73: 51-63Crossref PubMed Scopus (178) Google Scholar, 3Bargmann C.I. Cell. 1997; 90: 585-587Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 5Hille B. Neuron. 1992; 9: 187-195Abstract Full Text PDF PubMed Scopus (385) Google Scholar); thus, they have become prime targets for novel pharmacophores (1Savarese T. Fraser C. Biochem. J. 1992; 283: 1-19Crossref PubMed Scopus (441) Google Scholar, 4Intelligent Drug Design (1996) Nature 384, (suppl.) 5Google Scholar). In addition, congenital receptor dystrophies lead to a wide variety of functional heterogeneous disorders for which the molecular pathogenesis remains unknown (6Clapham D. Cell. 1993; 75: 1237-1239Abstract Full Text PDF PubMed Scopus (61) Google Scholar). Many of these mutations seem to affect the biogenesis of these receptors. Yet, the molecular components and mechanisms underlying the biogenesis of seven-transmembrane receptors have for the most part not been identified. The light receptors, opsins, constitute a class of homologous receptors that are key players in the activation of the photo-transduction cascade across species (7Okano T. Kojima D. Fukada Y. Shichida Y. Yoshizawa T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5932-5936Crossref PubMed Scopus (315) Google Scholar, 8Applebury M. Soc. Gen. Physiol. Ser. 1994; 49: 235-248PubMed Google Scholar, 9Pak W. Photobiochem. Photobiophys. 1986; 13: 229-244Google Scholar, 10Hargrave P. Hamm H. Hofmann K. Bioessays. 1993; 15: 43-50Crossref PubMed Scopus (77) Google Scholar). Opsins are continuously produced in large amounts in photoreceptors and constitute about 90% of the membrane protein content of the outer segments of vertebrate photoreceptors (11Molday R.S. Molday L.L. J. Cell Biol. 1987; 105: 2598-2601Crossref Scopus (108) Google Scholar). These cells likely express a highly efficient biogenic machinery to process and sort in a vectorial fashion the vast amounts of opsin produced in these cells. Thus, photoreceptors constitute an excellent model system to dissect the biogenic machinery of seven-transmembrane receptors. In Drosophila melanogaster, at least nine different gene products specifically affect the functional production of several opsin subclasses in photoreceptors (12Larrivee D.C. Conrad S. Stephenson R. Pak W.L. J. Gen. Physiol. 1981; 78: 521-545Crossref PubMed Scopus (70) Google Scholar, 13Stephenson R. O'Tousa J. Scavarda N. Randall L. Pak W.L. Cosens D.J. Vince-Price D. The Biology of Photoreception. Cambridge University Press, Cambridge1983: 447-501Google Scholar, 14Pak W.L. Invest. Ophthalmol. Vis. Sci. 1995; 36: 2340-2357PubMed Google Scholar). Among these, the ninaA gene encodes a retina-specific cyclophilin/peptidyl prolyl cis-transisomerase (PPIase) (15Shieh B.-H. Stamnes M. Seavello S. Harris G. Zuker C.S. Nature. 1989; 338: 67-70Crossref PubMed Scopus (215) Google Scholar, 16Schneuwly S. Shortridge R. Larrivee D. Ono T. Ozaki M. Pak W.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5390-5394Crossref PubMed Scopus (151) Google Scholar). The ninaA gene is expressed in all classes of photoreceptor cells (17Stamnes M. Shieh B.-H. Chuman L. Harris G. Zuker C.S. Cell. 1991; 65: 219-227Abstract Full Text PDF PubMed Scopus (221) Google Scholar), but mutations in this gene affect the production of only a subset of opsins, R1–6 opsin (12Larrivee D.C. Conrad S. Stephenson R. Pak W.L. J. Gen. Physiol. 1981; 78: 521-545Crossref PubMed Scopus (70) Google Scholar, 17Stamnes M. Shieh B.-H. Chuman L. Harris G. Zuker C.S. Cell. 1991; 65: 219-227Abstract Full Text PDF PubMed Scopus (221) Google Scholar). The molecular basis for the NinaA substrate specificity remains to be understood. We have identified in bovine retina a cyclophilin-related protein made up of multiple and well defined structural modules (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) that is the counterpart protein of the human and murine RanBP2 reported by the Nishimoto (19Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. Fukui M. Nishimoto T. Nature. 1995; 376: 184-188Crossref PubMed Scopus (411) Google Scholar), Coutavas (20Wu J. Manutis M. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), and Dabauvalle (21Wilken N. Senecal J.-L. Sheer U. Dabauvalle M.-C. Eur. J. Cell Biol. 1995; 68: 211-219PubMed Google Scholar) groups. Expression of RanBP2 is highly tissue-restricted in the retina (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) (albeit expression has been found in the liver (19Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. Fukui M. Nishimoto T. Nature. 1995; 376: 184-188Crossref PubMed Scopus (411) Google Scholar, 20Wu J. Manutis M. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar)) and present at high levels in cone photoreceptors (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We have shown that the C-terminal supradomain of RanBP2, Ran-binding domain 4 (RBD4)1 and cyclophilin (CY), mediate the selective association of RanBP2 with a subclass of opsins, red/green opsins (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar). This effect consists in the sequential, direct, and selective PPIase-dependent modification of opsin by CY, production of an RBD4-binding competent isoform of opsin, binding of modified opsin to the C-terminal half of RBD4, and, in concert with the N-terminal half domain of RBD4, CY-mediated chaperoning of the modified opsin isoform to the C-terminal half domain of RBD4 (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar). This leads to the production in vitro of a novel and immunoreactive opsin isoform with no propensity to self-aggregate and an increase in vivo of functional opsin receptor (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar). Based on our results, we have proposed that prolyl-isomerization constitutes a novel molecular switch in receptor biogenesis (23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar). This switch is modulated by the PPIase activity of cyclophilin required for the formation of a transient opsin isoform competent to be loaded onto component(s) of the processing and/or sorting machinery (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar). Recent evidence suggests that this molecular switch is widespread to other biological functions such as controlling cell-cycle division (24Yaffe M. Schutkowski M. Shen M. Zhou X. Stukenberg P. Rahfeld J. Xu J. Kuang J. Kirschner M. Fischer G. Cantley L. Lu K. Science. 1997; 278: 1957-1960Crossref PubMed Scopus (678) Google Scholar) and gene expression activity (25Leverson J. Ness S. Mol. Cell. 1998; 1: 203-211Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), possibly through PPIase-mediated conformational changes in substrate activity. RanBP2 is also a component of the Ran-GTPase cycle (26Rush M. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (92) Google Scholar, 27Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). Analysis of Saccharomyces cerevisiae mutants and other studies have implicated Ran-GTPase and many of its regulatory and putative effector components in a wide variety of pleiotropic functions ranging from nuclear protein import to nucleocytoplasmic export of mRNA (26Rush M. Drivas G. D'Eustachio P. Bioessays. 1996; 18: 103-112Crossref PubMed Scopus (92) Google Scholar, 27Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 28Kadowaki T. Goldfarb D. Spitz L. Tartakoff A. Ohno M. EMBO J. 1993; 12: 2929-2937Crossref PubMed Scopus (154) Google Scholar, 29Cheng Y. Dahlberg J. Lund E. Science. 1995; 267: 1807-1810Crossref PubMed Scopus (106) Google Scholar, 30Schlenstedt G. Saavedra C. Loeb J. Cole C. Silver P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 225-229Crossref PubMed Scopus (168) Google Scholar, 31Ren M. Villamarin A. Shih A. Coutavas E. Moore M. LoCurcio M. Clarke V. Oppenheim J. D'Eustachio P. Rush M. Mol. Cell. Biol. 1995; 15: 2117-2124Crossref PubMed Scopus (60) Google Scholar, 32Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar). Yet, there is no RanBP2 orthologue in the yeast genome (27Ohno M. Fornerod M. Mattaj I.W. Cell. 1998; 92: 327-336Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), and RanBP2 is highly expressed in terminally differentiated neuroretinal cells (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). RanBP2 has also been localized at cytoplasmic fibrils emanating from the nuclear pore complex (19Yokoyama N. Hayashi N. Seki T. Pante N. Ohba T. Nishii K. Kuma K. Hayashida T. Miyata T. Aebi U. Fukui M. Nishimoto T. Nature. 1995; 376: 184-188Crossref PubMed Scopus (411) Google Scholar, 20Wu J. Manutis M. Kraemer D. Blobel G. Coutavas E. J. Biol. Chem. 1995; 270: 14209-14213Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 21Wilken N. Senecal J.-L. Sheer U. Dabauvalle M.-C. Eur. J. Cell Biol. 1995; 68: 211-219PubMed Google Scholar), thus supporting the proposal that RanBP2 may play a key role in the nucleocytoplasmic transport of certain proteins and mRNAs. To better understand the RanBP2 role in cell function, it is of paramount importance to identify its molecular partners. To this end, we have extended structure-function studies to another RanBP2 domain, the cyclophilin-like domain (CLD) (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). We identified this domain as a region with low but significant homology to the C-terminal cyclophilin domain of RanBP2 (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The results reported here link RanBP2 to the proteasome proteolytic pathway and possible proteolytic processing of RanBP2. Moreover, this process may be mediated by another protein such as a putative and novel kinesin-like motor protein. We discuss the implication of these findings in protein biogenesis. Glutathione S-agarose beads, Coomassie stain, silver-stain kit, and Fast Stain concentrate were purchased from Amersham Pharmacia Biotech, Acros, Bio-Rad, and Zoion, respectively. Polyvinylidene difluoride membranes were purchased from Millipore and Applied Biosystems. Bovine retinas were purchased from Peel-Freeze (Roger, AR) and a local slaughter house. All nonhydrolyzable nucleotides, thrombin, and apyrase (grade VI, catalog number 6410) were purchased from Sigma. Mixture of protease inhibitors were from Boehringer Mannheim. Lactacystine and MG-132 were from BIOMOL. Protein markers (regular and prestained) were from New England Biolabs. Protein quantitation was carried out with Bio-Rad protein assay reagent. Crude retinal extracts were prepared exactly as described before (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar) and stored at −80 °C with the exception that tablets containing a mixture of protease inhibitors were added to the homogenization buffer as recommended by the manufacturer. When applicable, proteasome inhibitors, lactacystine and MG-132, were also added to the homogenization buffer at final concentrations of 20 nm and 20 μm, respectively. GST-RBD3-CLD-W1-W2, GST-CLD-W1-W2, GST-CLD, GST-RBD3-CLD, and GST-RBD3 constructs were prepared by subcloning, respectively, Klenow-treated SalI-BamHI,Sau96I-Sau96I, SalI-PvuII, and SalI-HphI fragments of CY15 clone (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) intoEcoRI and Klenow-treated pGEX-KG vector (33Guan K. Dixon J. Anal. Biochem. 1990; 192: 262-267Crossref Scopus (1640) Google Scholar). GST-W1-W2 protein construct was prepared by subcloning theBsu36I-BamHI of CY15 (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) into pGEX-KG digested with XhoI and treated with Klenow. Expression, purification, and concentration of GST-fused and thrombin-cleaved (unfused) constructs were carried out exactly as described before (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Purified GST-fused and unfused proteins were resolved on SDS-PAGE followed by Coomassie and/or silver-stain analysis. Incubation reactions of GST-fused constructs (2.2 μm) with bovine retinal extracts (80 μl, ∼2.5 mg of extract) and pull-down assays were carried out exactly as described previously (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar). When applicable, nonhydrolyzable nucleotides were added at a concentration of ∼1 mm and apyrase in the amount of 50 units. Unfused construct (competitor) was used at 10 fold molar excess. Coprecipitates were analyzed on 7.5% SDS-PAGE after boiling in SDS-sample buffer. P112 purification was carried out by scaling-up the incubation binding assays ∼180-fold between eight 15-ml conical tubes. The washings of coprecipitates were scaled-up accordingly. Coprecipitates were boiled in SDS-sample buffer for 3–5 min and loaded on 7.5% preparative SDS-PAGE, stained with Fast Stain followed by electroelution of P112 bands into Centricons. Eluted P112 protein was concentrated, and 5% (v/v) of the total concentrated protein was resolved in SDS-PAGE in parallel with coprecipitates of analytical retinal binding reactions and analyzed by silver-stain. P99 purification procedure was carried out exactly the same way with the exception that incubation assays were scaled-up ∼360-fold, and 20% (v/v) of concentrated eluted protein was loaded on SDS-PAGE for silver-stain analysis. The rest of purified proteins were loaded on SDS-PAGE, blotted onto PVDF membranes, stained for 1–2 min with 0.1% Coomassie Blue in 50% methanol and 1% acetic acid, washed several times with destaining solution (50% methanol, 1% acetic acid), followed by several washes with Millipore H2O. The blue-stained bands were cut and subjected to Edman degradation and amino acid composition analysis. Incubation reactions and pull-down assays were carried out as described before. GST-CLD coprecipitates were resolved on SDS-PAGE, blotted onto PVDF membranes, blocked, and incubated with PA700 antibody at 1:2,500 (gift from Dr. George DeMartino) by the same exact procedures previously described (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar) with the exception that IgY secondary antibody (Promega) and a more sensitive chemiluminescent substrate (Super Signal, Pierce) were used for the development of the blot. Bovine retinal extracts (150 μg) were loaded and resolved on SDS-PAGE, blotted onto PVDF membranes, blocked, and incubated with Cy321 antibody (1:5,000) by the same exact procedures previously described (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar) with the exception of the use of a more sensitive chemiluminescent substrate (Super Signal, Pierce). We continued the structure-function analysis of RanBP2 by the same exact methods we previously reported (22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar). To this end, we prepared a series of GST fused and unfused protein constructs containing the whole RBD3, CLD, W1, and W2 supradomain and several fragments thereof (Fig. 1). The GST-fused constructs were incubated with CHAPS-solubilized retinal extracts, and pull-down assays were carried out in search for coprecipitating proteins with binding activity to the bait proteins. Coprecipitating proteins were subjected to several washes followed by SDS-PAGE and silver-stain analysis. To test the specificity of the binding of retinal proteins to GST-fused constructs, free (unfused) bait was added to the incubation reactions to compete with the GST-fused construct for the target substrate (Fig. 2 a, lane 4). In certain reactions, nonhydrolyzable nucleotide analogs were also added to test for nucleotide-dependent association of retinal substrate(s) with the bait moiety (Fig. 2 a, lane 5).Figure 2Identification of retinal CLD binding proteins. a, Silver-stain SDS-PAGE analysis of glutathione S-agarose coprecipitates of incubation reactions with GST-CLD. Incubation of GST-CLD with retinal extracts in the presence (lane 7) and absence (lane 2) of proteasome inhibitors lead to the coprecipitation of several retinal protein species. Among these, only P112 and P99 specifically coprecipitated with GST-CLD because addition of unfused CLD to the reactions abolished their association with GST-CLD (lane 3). Addition of ATPγS (lane 4) as well as GTPγS (not shown) and apyrase (lane 5) to the binding reactions resulted, respectively, in a decrease and no change of binding of P112 and P99 to GST-CLD. Addition of ATPγS to the incubation reaction also abolished the binding and lead to the association, respectively, of 70- and 68-kDa proteins. However, this effect was nonspecific, as it was observed also for other unrelated GST-fusion constructs (not shown).Lane 2 represents an aliquot (15 μg) of purified GST-CLD (asterisk) used in incubation reactions. The bands below GST-CLD are lower molecular weight degradation products of GST-CLD. b, retina-selective association of P112 and P99 with CLD of RanBP2. P112 and P99 did not coprecipitate with GST-CLD after incubation with liver (lane 2), kidney (lane 3), and spleen (lane 4) extracts. Concentration of tissue extracts were normalized to those of retinal extracts.Lane 1, protein markers; asterisk, GST-CLD; R.E. and R.E.*, retinal extracts prepared, respectively, in the presence of mixture of protease inhibitors and, in addition, the proteasome inhibitors, lactacystine and MG-132; GST-CLD, GST-fused CLD; CLD, free (unfused) CLD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast to RBD4-CY (18Ferreira P. Hom J. Pak W. J. Biol. Chem. 1995; 270: 23179-23188Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 22Ferreira P. Nakayama T. Pak W. Travis G. Nature. 1996; 383: 637-640Crossref PubMed Scopus (188) Google Scholar, 23Ferreira P. Nakayama T. Travis G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1556-1561Crossref PubMed Scopus (49) Google Scholar), the large GST-fused construct containing RBD3, CLD, and W1 and W2 in the same polypeptide chain was highly unstable when expressed and purified from Escherichia coli (not shown). Thus, we expressed these domains singly to facilitate SDS-PAGE analysis of binding activity of retinal substrates to GST constructs. In this study, we searched for retinal substrates that specifically associated with the CLD domain of RanBP2. Incubation of GST-CLD with retinal extracts under physiological conditions lead to the association to this construct of two proteins with an apparent molecular mass of 112 kDa, P112 (Fig. 2 a, arrow, lane 3), and 99 kDa, P99 (Fig. 2 a, filled arrowhead, lane 3). Binding of these proteins to the CLD moiety of GST-CLD was highly specific because these were the only two proteins whose association with the GST-construct could be disrupted by coincubation with free (unfused) CLD (Fig. 2 a, lane 4). This also showed that the 112- and 99-kDa bands represented single protein species. Moreover, these proteins did not bind other unrelated GST-fusion constructs such as GST-W1-W2 (not shown) and GST-RBD3 (not shown). P112 had a much higher binding activity toward CLD than P99, suggesting that P99 is specifically copurifying with P112. Association of P112 and P99 with CLD was reduced in the presence of ATPγS (Fig. 2 a, lane 5), but it was not affected by the presence of the ATP-degrading enzyme, apyrase (Fig. 2 a, lane 6), GTPγS, and GDPβS (not shown). Finally, incubation reactions carried out with retinal extracts prepared in the presence of the proteasome inhibitors (34Rock K. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2196) Google Scholar,35Palombella V. Rando O. Goldberg A. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar), lactacystine and MG-132, did not alter significantly the binding activity of these proteins to CLD (Fig. 2 a, lane 7). To investigate if the association of P112 and P99 with CLD was selective for retinal extracts, we incubated GST-CLD exactly under the same conditions with three different extracts prepared from bovine liver, kidney, and spleen tissues. The protein concentration of these were normalized to the same exact concentration as that for retinal extracts. To this end, binding activity of P112 and P99 to CLD was not detected with these tissue extracts (Fig. 2 b). To determine the identity of CLD-binding P112 and P99 proteins, we performed large scale retinal incubation reactions with GST-CLD. To this end, analytical incubation reactions were scaled-up approximately 180- and 360-fold for the purification of P112 and P99, respectively. GST-CLD coprecipitating proteins from large scale preparation reactions were loaded on large preparative SDS-PAGE, and retinal P112 and P99 were electroeluted and concentrated. To confirm the isolation and purity of the CLD-binding proteins, 5% (v/v) of the total amount of purified protein was run side-by-side in SDS-PAGE with analytical incubation reactions as well as with the remaining bulk of the purified protein. As seen in Figs. 3 a and 5 a, respectively, purified P112 and P99 comigrated exactly with those resolved from coprecipitates of analytical incubation reactions, albeit some lower molecular weight species were observed as a result of some degradation of the purified proteins.Figure 5Purification of retinal P99. a, retinal incubation reactions with GST-CLD were scaled-up, the P99 band was electroeluted and concentrated, and an aliquot was loaded on an SDS-PAGE gel for silver-stain analysis (lane 2) in parallel with analytical retinal incubation reactions (lanes 3–5) as described in Fig. 2 a. Purified P99 (arrow) migrated exactly at the same position (double-headed arrow) as the specific GST-CLD coprecipitating protein, P99, observed in analytical incubation reactions (lane 4). Some degradation of P99 (arrowhead) into P97 (open arrowhead) was observed. b, Coomassie Blue-stained PVDF blot of purified P99. P99 protein (arrowhead) and a slightly smaller proteolytic product of P99, P97 (open arrowhead), purified from retinal extracts were blotted onto a PVDF membrane and stained with Coomassie Blue before being subjected to Edman degradation analysis. P99 N terminus was blocked while the proteolytic product, P97, provided N-terminal sequence information (Fig. 6).Arrowhead, purified GST-CLD co-precipitating protein of 99 kDa; open arrowhead, P97 proteolytic product of P99.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To ascertain the identity of P112, this purified protein was blotted onto PVDF membranes, and the 112-kDa band (Fig. 3) was subjected to Edman degradation. N-terminal sequencing of the first 19 amino acids of P112 showed 100% identity to the counterpart sequence of the bovine (36DeMartino G. Moomaw C. Zagnitko O. Proske R. Chu-Ping M. Afendis S. Swaffield J. Slaughter C. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar) and human (37Yokota K. Kagawa S. Shimizu Y. Akioka H. Tsurumi C. Noda C. Fujimuro M. Yokosawa H. Fujiwara T. Takahashi E. Ohba M. Yamasaki M. DeMartino G. Slaughter C. Toh-e A. Tanaka K. Mol. Biol. Cell. 1996; 7: 853-870Crossref PubMed Scopus (54) Google Scholar) P112 subunits of the 19 S regulatory complex of the 26 S proteasome (Fig. 4). N-terminal sequencin

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