Prefoldin-nascent chain complexes in the folding of cytoskeletal proteins

Abstract

In vitro transcription/translation of actin cDNA and analysis of the translation products by native-PAGE was used to study the maturation pathway of actin. During the course of actin synthesis, several distinct actin-containing species were observed and the composition of each determined by immunological procedures. After synthesis of the first approximately 145 amino acids, the nascent ribosome-associated actin chain binds to the recently identified heteromeric chaperone protein, prefoldin (PFD). PFD remains bound to the relatively unfolded actin polypeptide until its posttranslational delivery to cytosolic chaperonin (CCT). We show that alpha- and beta-tubulin follow a similar maturation pathway, but to date find no evidence for an interaction between PFD and several noncytoskeletal proteins. We conclude that PFD functions by selectively targeting nascent actin and tubulin chains pending their transfer to CCT for final folding and/or assembly.

Figure 3

Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [³⁵S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [³⁵S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [³⁵S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.

Figure 1

Intermediates in the biogenesis of full-length β-actin. (A) The reticulocyte lysate was programmed with full-length mRNA encoding β-actin in the presence of [³⁵S]methionine. Edeine (10 mM) and 7-^MeGMP (4 mM) were added after 12 min to block new initiation of translation. Aliquots were removed at various times (indicated at the top of the panel), the samples incubated with apyrase (to deplete ATP) and puromycin (to release nascent chains), and analyzed by native-PAGE (top). The migration of native molecular mass markers is shown on the left. The positions of migration of actin monomer along with four other actin-containing species (I, II, III, and IV) are shown on the right. The same samples, analyzed by SDS-PAGE, are shown in the lower panel. The amount of total material applied to the native gel is twice that loaded on the SDS-PAGE. Fluorograms of the gels are shown after 2.5 h of exposure. (B) Kinetics of appearance of the species detected after native-PAGE (species I, II, III, and monomeric actin). The optical density of each band from the x-ray film was calculated with NIH Image software. The optical density units derived for each species are plotted as a function of time. (C) Kinetics of appearance of the species detected after native-PAGE, plotted as follows: optical density data from B for each species were divided by the sum of the optical density units of all the radiolabeled species recovered in the gel for a given time point. The percentage of each actin species (I, II, III, and monomeric actin) is plotted as a function of time. (D) To examine the earliest forms of nascent actin, actin mRNA was translated for 2 min, and then edeine and 7-^MeGMP were added. Aliquots were removed every 2 min and the actin-containing complexes fractionated by native-PAGE. The percentages of actin chains in species I or II were determined as described in C.

Figure 2

COOH-terminal truncated actin accumulates as the species I complex. (A) The reticulocyte lysate was programmed with mRNA encoding a 336–amino acid NH₂-terminal fragment of actin (full-length actin is 374 amino acids) and the reaction products analyzed as in Fig. 1 A. The amount of total material applied to the native-PAGE (top) is twice that of the amount loaded on the SDS-PAGE (bottom). Fluorograms of the gels are shown after 2.5 h of exposure. The migration of native molecular mass markers is shown on the left and the actin species shown at the right. (B) Kinetics of appearance of the species detected after native-PAGE was plotted as follows: optical density value derived for each species was divided by the sum of the optical density units of all the radiolabeled species recovered in the gel for a given time point. The percentage of each actin species (I, II, and monomeric actin) is plotted as a function of time. (C) In vitro translation products of either full-length actin mRNA (Actin), or the 336–amino acid actin truncation mRNA (Actin/336) were analyzed in the first dimension by native-PAGE. The lane containing the reaction material was excised and then analyzed by SDS-PAGE as described in Materials and Methods. The starting materials, actin/336 or full-length actin, were loaded in lanes 1 and 2, respectively (left). Fluorograms of the gels are shown.

Figure 4

Native-PAGE analysis of different COOH-terminal truncated actin polypeptides. Reticulocyte lysate was programmed with either full-length actin mRNA or a number of progressively shorter mRNA species encoding COOH-terminal deletions. The predicted number of amino acids translated is shown. Translations were initiated in the presence of [³⁵S]methionine, and then edeine and 7-^MeGMP were added to block the initiation of new chains at different times (see Materials and Methods). Aliquots were taken either early (e) or later (l) after edeine and 7-^MeGMP addition, to visualize the different species of nascent actin polypeptides. Note that for the SDS-PAGE analysis (A) each reaction was divided: one half was untreated (−), while puromycin and RNase were added to the other half to release nascent chains (+). Products were analyzed by SDS-PAGE. Analysis of reactions via native-PAGE (B), puromycin was added to each of the samples and the material applied to the gel. Shown are fluorographs of the gels, with molecular mass markers indicated on the left. The positions of the different actin species, as well as monomeric actin, are shown at the right in B.

Figure 5

The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH₂-terminal fragment of actin. After 8 min, edeine and 7-^MeGMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.

Figure 6

Actin bound to PFD is nonnative. (A) Actin purified from rabbit muscle (pure), along with either in vitro translated [³⁵S]methionine-labeled full-length actin (FL) or the 336–amino acid NH₂-terminal fragment of actin (336) were each incubated at 23°C for 30 min, under control conditions (i.e., no added protease, indicated as in the panel, lanes 1, 4, and 5); with 0.15 mM of trypsin (T, lanes 2, 6, and 8) or 0.375 mM of trypsin (T, lanes 3, 7, and 9); with 0.6 mM of chymotrypsin (C, lanes 10, 12, and 14) or 1.5 mM of chymotrypsin (C, lanes 11, 13, and 15). Digestions were terminated by addition of 1 mM PMSF and the products were analyzed by SDS-PAGE. Shown is the Coomassie blue stained image of the purified actin, and a fluorograph of the gel containing the in vitro translated substrates. (B) Full-length [³⁵S]methionine-labeled actin was synthesized in bacteria and then purified by anion exchange chromatography on MonoQ. A portion of the purified radiolabeled full-length recombinant actin was denatured by treatment with 8 M urea, and then was diluted out of urea in the presence of purified PFD. The recombinant actin–PFD complex was purified by gel filtration. Both the native actin (left) and the actin–PFD complex (right) then were incubated at 25°C with 50 nM proteinase K for the times indicated. The reactions were terminated by the addition of Laemmli sample buffer and the products resolved by SDS-PAGE. A fluorogram of the gel is shown.

Figure 7

The maturation pathway of β-tubulin involves both PFD and CCT. (A) Reticulocyte lysate was programmed with full-length mRNA encoding chicken β-tubulin in the presence of [³⁵S]methionine. Edeine and 7-^MeGMP were added after 8 min to block new initiation of translation. Aliquots of the reaction mixture were removed at various times (indicated at the top of the panel), the samples incubated with apyrase and puromycin, and analyzed by native-PAGE (top). On the right, the positions of migration of the tubulin–PFD complex and the β-tubulin–CCT complex are indicated. An open arrowhead marks the position of a fast migrating β-tubulin containing product (likely to be α/β-tubulin heterodimer, β-tubulin–cofactor A complex, or a mixture of the two). The filled arrowhead indicates the position of migration of a species that occurs very late in time, which likely is β-tubulin complexed with one of the several tubulin-specific chaperones that facilitate tubulin heterodimer formation. (B) Analysis of the same samples shown in A by SDS-PAGE. (C) Kinetics of appearance of species detected by native-PAGE in A was determined as described for Figs. 1 and 2. (D) Puromycin-released β-tubulin chains produced after 18 min of translation (shown in A) were incubated at 24°C for 15 min with preimmune serum, anti–PFD 6 serum, or purified anti–TCP-1α monoclonal IgG 23C, and the reaction products were analyzed by native-PAGE. The positions of tubulin bound to CCT and tubulin bound to PFD are indicated on the left.

Figure 8

Cytoskeletal proteins, actin, and tubulin interact with PFD and then with CCT during the course of their maturation in vivo. Data presented here indicate that at least a portion of actin, as well as α- and β-tubulin, interacts with the PFD chaperone while still bound to the ribosome. Following release from the ribosome, the full-length newly synthesized actin and tubulins are transferred over to the CCT for completion of folding to the native state. It remains possible that the actin or tubulin molecules that fail to reach the native state upon release from the chaperonin rebind to PFD and perhaps are transferred to the chaperonin again to allow for a new round of folding. Finally, full-length native actin, which is denatured, likely interacts with PFD before being transferred over to the CCT (Vainberg et al., 1998). It remains possible that other pathways of actin maturation may also exist in the living cell.