Regulatory Interaction of Phosducin-Like Protein with the Cytosolic Chaperonin Complex

Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex Joseph N. McLaughlin*, Craig D. Thulin*, Sarah J. Hart*, Katheryn A. Resing†, Natalie G. Ahn†‡, and Barry M. Willardson*§ *Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602; and †Department of Chemistry and Biochemistry and ‡Howard Hughes Medical Institute, University of Colorado, Boulder, CO 80309 Edited by Melvin I. Simon, California Institute of Technology, Pasadena, CA, and approved April 9, 2002 (received for review February 7, 2002) Phosducin and phosducin-like protein (PhLP) bind G protein ␤␥ sub- a high-affinity in vivo interaction of PhLP with CCT that inhibits the units and regulate their activity. This report describes a previously ability of CCT to catalyze the folding of its protein substrates. uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identiﬁ- Materials and Methods cation. Chaperonin containing tailless complex polypeptide 1 (CCT), a cDNA Constructs. Rat PhLP cDNA was cloned into the vector cytosolic chaperone responsible for the folding of many cellular pcDNA3.1(ϩ)͞myc-His B (Invitrogen) to generate a construct with proteins, binds PhLP with a stoichiometry of one PhLP per CCT a c-myc epitope tag and a hexahistidine purification tag on the 3Ј complex. Unlike protein-folding substrates of CCT, which interact only end. The resulting PhLP-myc-His cDNA was cloned into the vector in their nonnative conformations, PhLP binds in its native state. Native pET15b (Novagen) for expression and purification. Rat Pd-myc- ϩ PhLP competes directly for binding of protein substrates of CCT and His was also prepared in pcDNA3.1( ) and pET15b. N-terminally thereby inhibits CCT activity. Overexpression of PhLP inhibited the tagged His-PhLP in pET15b was prepared as described (25). The ability of CCT to fold newly synthesized ␤-actin by 80%. These results integrity of all constructs was verified by sequence analysis. A ␤ ϩ ͞ ͞ suggest that the interaction between PhLP and CCT may be a means -galactosidase-myc-His vector pcDNA3.1( ) myc-His LacZ was to regulate CCT-dependent protein folding or alternatively, to control obtained from Invitrogen. the availability of PhLP to modulate G protein signaling. Protein Purifications. PhLP-myc-His was expressed in Escherichia coli and purified by Ni2ϩ affinity chromatography in the absence of lthough all of the necessary information for achieving a specific denaturant as described (25). 35S-labeled His-PhLP was expressed Athree-dimensional structure is contained within a protein’s and purified in a similar manner with minor modifications. E. coli primary sequence, many proteins require interaction with chaper- DE3 cells were grown in M9 minimal medium, and 10 ␮Ci/ml 35 ϭ ones to attain their native conformation in vivo. One class of [ S]H2SO4 (NEN-Dupont; 1 Ci 37 GBq) was added at the time chaperones, the eukaryotic cytosolic chaperonin CCT [chaperonin of induction of His-PhLP expression to label the protein. The containing tailless complex polypeptide 1 (TCP-1)] consists of eight 35S-His-PhLP was insoluble in cells grown in minimal media, homologous polypeptides that form a ring-shaped structure with a therefore the cell pellets were extracted in buffer with8Mureaand ϩ large central cavity (1). Two of these rings are stacked together to applied to the Ni2 -chelate column. The 35S-His-PhLP was rena- make the holo-CCT complex that uses the energy derived from tured on the column by washing in buffer containing decreasing ATP binding and hydrolysis to drive the folding of proteins in the amounts of urea and then eluted in its refolded form with imidazole. central cavity in an enzyme͞substrate fashion (1–5). CCT assists in 35S-labeled Pd was prepared in a similar manner except that urea ␣ the folding of actin and tubulin (2, 6, 7) and many other cytosolic was not needed to solubilize the protein. Gt (15), Hsp 70 (26), and ␣ proteins (3, 4, 8). In fact, CCT interacts with both nonnative CCT (27) were each purified following described protocols. Gt was conformations of proteins and nascent polypeptide strands (9). labeled with 125I, also as described (28). There has been extensive study of the mechanism of protein folding Immunoprecipitation. Chinese hamster ovary (CHO) cells were by CCT (1–5), yet little is known about the cellular regulation of this grown in 6-well plates to 90% confluency. Cells were transfected process. In this report, we describe an interaction between CCT and with 1 ␮g DNA per well by using 2 ␮g of Lipofectamine Plus reagent a native polypeptide, phosducin-like protein (PhLP), which blocks (GIBCO͞BRL) according to the manufacturer’s protocols. Two the ability of CCT to fold actin and other substrates. days (48 h) later, cells from each well were washed twice in ice-cold PhLP, so named for its 65% homology in amino acid sequence PBS and lysed in 200 ␮l of immunoprecipitation buffer [20 mM to phosducin (Pd), was discovered as the product of an ethanol- ͞ ͞ ͞ ␮ NaH2PO4,pH7.4150 mM NaCl 2% IGEPAL (Sigma) 8 M induced gene in cultured neuronal cells (10). Known functions of Pd E-64 (Boehringer Mannheim)͞0.6 mM PMSF]. Lysates were and PhLP involve regulation of G protein signaling. Pd binds the ␤␥ ␣ passed seven times through a 25-gauge needle and centrifuged at G subunit complex and blocks its interaction with G subunits 11,000 ϫ g for 10 min. Tagged PhLP was immunoprecipitated with as well as G␤␥ effectors (11–15). PhLP also binds G␤␥ (16, 17) and ␮ ␮ ␮ ␤␥ ␣ 4 lof0.7 g/ l anti-c-myc antibody (BioMol, Plymouth Meeting, prevents G from binding G (18). In addition, both Pd and PhLP PA) for 30 min at 4°C and 20 ␮l of a 50% slurry of Protein A͞G interact with the 26S proteasomal subunit, SUG1 (19, 20). How- PLUS agarose (Santa Cruz Biotechnology) for an additional 30 min ever, their cellular expression patterns are very different. Pd is at 4°C. Samples were centrifuged, and the beads were washed three expressed almost exclusively in retinal and pineal cells (21, 22), times with 300 ␮l of immunoprecipitation buffer. The pellet was whereas PhLP mRNA has been found in all tissues examined (10, solubilized in Laemmli SDS͞PAGE sample buffer, separated on a 23), and immunoblot analyses has confirmed PhLP protein expres- 12% Laemmli SDS͞PAGE gel, and stained with GelCode Coo- sion in diverse tissues and cell lines (C.D.T., unpublished data). As massie Reagent (Pierce) or immunoblotted by using a 1:50,000 a result of its broad expression pattern, PhLP has been postulated dilution of anti-PhLP antiserum (18), a 1:10,000 dilution of anti-Pd to be a general regulator of G protein signaling (17). However, a recent study in yeast suggests PhLP involvement in other cellular processes (24). In an effort to better understand the cellular This paper was submitted directly (Track II) to the PNAS ofﬁce. function of PhLP, we sought to identify its in vivo binding partners Abbreviations: TCP-1, tailless complex polypeptide 1; CCT, chaperonin containing TCP-1; PhLP, by coupling coimmunoprecipitation techniques with mass spectro- phosducin-like protein; Pd, phosducin; CHO, Chinese hamster ovary. metric identification of coimmunoprecipitating proteins. We report §To whom reprint requests should be addressed. E-mail: barry࿝[email protected]. 7962–7967 ͉ PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.112075699 Downloaded by guest on September 26, 2021 antiserum (18), or a 1:500 dilution of anti-TCP-1␣ (Calbiochem), ␤, Luciferase activity was measured according to the manufacturer’s or ␧ antiserum (Stressgen) as primary antibodies. Blots were protocol (PharMingen) at increasing times after the addition of developed by using ECLϩ (Amersham Pharmacia) and were reticulocyte lysate. From these data, the initial rates of luciferase visualized on a Storm 860 PhosphorImager (Molecular Dynamics) refolding were determined. No increase in basal luciferase activity in chemiluminescence mode. Bands were quantified by using Im- with time was observed in the absence of reticulocyte lysate. ageQuant software (Molecular Dynamics) and the ratio of PhLP to In Vivo ␤-Actin Folding. The procedure followed described methods CCT in the immunoprecipitate was determined by comparing the for measuring CCT-dependent actin folding in cells (3). CHO cells chemiluminescence from known amounts of purified PhLP or CCT were transiently transfected as before with PhLP or LacZ in the with that from the immunoprecipitates. Coimmunoprecipitation of pcDNA3.1(ϩ)B vector. Cells were exposed to 35S-Met (NEN- endogenous PhLP with CCT was performed in a similar manner Dupont) for 10 min. Protein synthesis was stopped by addition of but with untransfected CHO cells and 4 ␮lof1␮g/␮l anti-TCP-1␣ 4 ␮M cycloheximide (ICN) and the incubation was continued an antibody as the precipitating antibody. additional 5 min. Cells were washed three times with PBS to remove For in vitro immunoprecipitations from human cell lines, cell free 35S-Met and were lysed in 20 mM Hepes, pH 7.4͞50 mM KCl͞1 lysates were prepared as described above. After clarifying by mM DTT͞0.2 mM CaCl ͞4 mM cycloheximide͞1 mM PMSF͞0.5 centrifugation, 10 ␮g of purified PhLP-myc-His was added and 2 ␮g/ml leupeptin͞0 ␮M pepstatin͞0.4 units/␮l hexokinase (Sig- incubated 30 min at 4°C. Immunoprecipitation was as described ma)͞40 mM glucose by passing through a 25-gauge needle several above. times. Cellular debris was removed by centrifugation, and the For in vitro immunoprecipitations from rabbit reticulocyte lysate, ␮ 125 ␣ 35 35 supernatants were incubated for1hinthepresence of DNase 30 M I-Gt , S-His-PhLP, or S-His-Pd was diluted 1:6 in I-affigel beads prepared as described (33).

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