Overexpression of wild-type and compound heterozygous HRD mutant forms of TBCE in HeLa cells. HeLa cells were transfected with constructs engineered for the expression of wild-type, CΔ, or deletion of nucleotides 66–67 (NΔ) mutant forms of TBCE. Cells were fixed 24 h posttransfection and analyzed by indirect immunofluorescence by using antisera for α-tubulin (shown in red) and TBCE (ref. 6; shown in green). Arrows highlight TBCE-expressing cells showing microtubule obliteration as a result of expression of the transgene. (Scale bar, 10 μm.)

Expression and stability of wild-type and HRD mutant forms of TBCE. (A) A cloned full-length TBCE cDNA sequence containing the NΔ deletion was altered by site-directed mutagenesis to change pairs of methionine codons (shown in turquoise at positions 3′, 12′, and 15′ in Fig. 3A) upstream of the genetic lesion to alanine codons (GCG), thus leaving only a single AUG triplet as a potential initiation codon. A construct (−M), in which all three AUG codons were altered to GCG, was also generated as a control. Each plasmid was used to transfect CHO K1 cells together with a plasmid engineered for the expression of GFP as a marker for transfection efficiency. Twenty-four hours posttransfection, cell extracts were prepared, and aliquots containing equal amounts of total protein were analyzed by Western blotting. (B) Analysis by Western blotting of CHO cells transfected with constructs with the NΔ deletion containing either an intact (lane 1) or an altered (ATG→TAG; lane 2) authentic initiation codon. (Lower) Shown is detection of cotransfected GFP as a control for transfection efficiency. (C) In vitro chaperonin-mediated β-tubulin-folding assays done with purified CCT, TBCC, and TBCD and supplemented with soluble extracts from wild-type or compound heterozygous HRD patient cells. Reaction products were resolved by nondenaturing PAGE and detected by autoradiography. Arrows (top to bottom) denote the location of the CCT/β-tubulin binary complex, the β-tubulin/TBCD complex, native tubulin heterodimers, and the complex formed between CCT-discharged β-tubulin-folding intermediates and TBCA contained in the cell extracts (5, 6). (D) Sensitivity to proteolysis of wild-type (w.t.) and HRD mutant (NΔ) forms of TBCE. Proteins were ³⁵S-labeled by coupled transcription/translation and incubated with a soluble extract prepared from HRD patient-derived fibroblasts for the times shown. The reaction products were analyzed by SDS/PAGE. Arrows denote location of intact proteins. (E) Half life of wild-type and cryptically initiated TBCE in vivo. HEK293 cells transfected with constructs engineered for the expression of wild-type and cryptically initiated (NΔ) TBCE were treated with cycloheximide and their TBCE content measured by Western blotting at the times shown. (F) Semiquantitative Western blot analysis of the tubulin content in normal and compound heterozygous HRD patient-derived fibroblasts. The blot is identical to that shown in Fig. 1C. Loading controls (done with an anti-β-actin antibody) are shown below. (G) Expression levels of TBCE in normal (w.t.) and compound heterozygous HRD patient cells determined by using an anti-TBCE antibody affinity-purified by using the cryptically initiated HRD NΔ protein expressed in E. coli as the selecting antigen. Use of this antibody avoids bias in favor of the wild-type protein as a result of potential recognition of epitopes in the wild-type protein that lie N-terminal to the genetic lesion. Loading controls (using an anti RhoGDI antibody) are shown below.

Expression and activity of wild-type and HRD mutant TBCE proteins. (A) Schematic representation of the tubulin heterodimer assembly pathway in higher eukaryotes (6, 9). (B) Expression levels of TBCE mRNA (relative to β-actin) in normal (w.t.) and compound heterozygote HRD patient-derived fibroblasts, determined by real-time PCR. (C) Abundance of TBCE protein determined by semiquantitative Western blotting by using an anti-TBCE antibody in postnuclear extracts of wild-type and HRD mutant (compound heterozygote) cells. Loading controls done with anti-β-actin and anti-RhoGDI antibodies are shown below. (D). Expression of wild-type (w.t.) and HRD mutant (CΔ, C371X; NΔ, deletion of nucleotides 66–67) forms of TBCE by transcription/translation in rabbit reticulocyte lysate. (E) Purification of recombinant wild-type and HRD mutant forms of TBCE expressed in insect sf9 cells. In D and E, location of molecular mass markers is shown on the left. (F) In vitro β-tubulin-folding assays (5, 6), in which bacterially expressed ³⁵S-labeled urea-unfolded β-tubulin was suddenly diluted into a reaction containing the cytosolic chaperonin CCT, ATP, GTP, TBCC, and TBCD and either wild-type (w.t.) or compound heterozygous HRD mutant forms (NΔ, deletion of nucleotides 66–67) of TBCE. Reaction products were analyzed by nondenaturing gel electrophoresis. Arrows (top to bottom) denote the location of the CCT/β-tubulin binary complex, the β-tubulin/TBCD complex, and native tubulin heterodimers. (G) Relative tubulin GAP-enhancing activities (17) of compound heterozygous HRD mutant [C371X (CΔ) and deletion of nucleotides 66–67 (NΔ)] forms of TBCE protein relative to wild type (taken as 100%).

The NΔ TBCE HRD allele is expressed as a result of translational initiation at three upstream out-of-frame AUG codons. (A) Nucleotide and corresponding amino acid sequences of wild-type TBCE (Upper) and the allele with the HRD deletion of nucleotides 66–67 (Lower) in the vicinity of the 2-bp deletion. Deleted nucleotides in HRD are shown (Upper) in red. Potential translational reading frames in the mutant HRD allele are shaded in gray and yellow. Numbering of amino acids in the −2 reading frame (1′, 2′, etc.) starts at the first residue (Q) of this open frame preceding the 2-bp deletion. Three M residues in this reading frame are highlighted in turquoise. Note that translational initiation at these methionines in the NΔ HRD allele leads to a transition to translation from the correct ORF, whereas translation initiated at the “correct” methionine in the mutant sequence (amino acids highlighted in brown) leads to termination at a stop codon located at nucleotides 148–150 (not shown). A polypeptide (MENMQLR) identified by mass spectroscopy in a tryptic digest of the protein expressed from the mutant HRD allele (see Fig. 6) is boxed. (B) Translational analysis of the NΔ HRD allele by site-directed mutagenesis. The mutated forms shown (corresponding triplets altered to alanine or termination codons are highlighted in A in pink and blue, respectively) were expressed as ³⁵S-labeled proteins by coupled transcription/translation in rabbit reticulocyte lysate and the reaction products detected by autoradiography following resolution by SDS/PAGE.

Schematic illustration (not drawn to scale) of cryptic out-of-frame translational initiation of mutant (deletion of nucleotides 66–67) TBCE. M1, authentic initiator methionine codon leading to translation of the wild-type sequence. M3′, M12′, and M15′ denote three AUG codons in the −2 reading frame (see Fig. 3A), each of which serves to initiate translation of the mutant allele. Note the transition of all three out-of-frame nonsense translation products (shown as jagged lines) to the correct ORF at the point of mutation (marked by a triangle). Initiation at the authentic M1 AUG codon in the mutant allele leads to translation (not shown) of a peptide consisting of the first 22 aa of TBCE fused to 26 nonsense amino acids translated from the −1 reading frame downstream of the mutation.