Plasmid Construction and Manipulation
Plasmids were engineered to enable the visualization of co-translational and post-translational protein interactions. This intricate process involved constructing sequences encoding human β-actin with various tags. These tags included 3×Flag and 24×MS2 stem-loop repeats (e.g., β-actin-24×MS2), as well as 10×SunTag repeats (e.g., β-actin-10×SunTag).
Plasmids were engineered to enable the visualization of co-translational and post-translational protein interactions.
Further variations included the generation of truncated versions of β-actin, alongside a G150P point mutation variant. Additional plasmids were prepared for the MS2 coat protein fused to GFP (MCP–GFP), MoonTag epitopes, VCP with HaloTag, and CCT4 with SNAP-tag. Control plasmids, such as a SunTag without β-actin, were also meticulously created.
Cell Line Development
Human U-2 OS cells were maintained under standard culture conditions. Stable cell lines were subsequently generated using diverse methodologies to express specific tagged proteins or achieve gene knockdowns.
Stable cell lines were generated using various methods to express specific tagged proteins or achieve gene knockdowns.
CRISPR–Cas9-mediated gene integration was employed to create U-2 OS cell lines expressing CCT4–HaloTag, PFD4–Halo, and PFD4–SNAP. Lentivirus transduction was utilized to establish a polyclonal U-2 OS line expressing RPL10A–SNAP-tag. Furthermore, a PFD3 knockdown stable cell line was established via CRISPR–Cas9-mediated gene deletion. All developed cell lines underwent routine testing to ensure the absence of mycoplasma contamination.
Biochemical Assays
Multiple biochemical techniques were utilized for comprehensive protein analysis, ensuring a thorough characterization of molecular interactions and kinetics.
SDS–PAGE and Immunoblotting
Proteins were separated by electrophoresis, transferred to PVDF membranes, and detected using specific primary and HRP-conjugated secondary antibodies. Quantification of protein levels was performed using Fiji.
HaloTag Pulldown
Cells were lysed, and tagged proteins were affinity-purified using Halo-Trap Magnetic Agarose beads. The eluted proteins were subsequently used for immunoblotting or further mass spectrometry analysis.
Kinetics of Actin Transit to TRiC
Puromycin chase experiments were performed on cells expressing actin–SunTag–Xbp1u+ variants to precisely determine the folding half-time of actin. Data were fitted to an exponential decay function to derive kinetic parameters.
Puromycin chase experiments were performed to determine the folding half-time of actin.
Cycloheximide Chase
Cells were treated with cycloheximide (CHX) to inhibit protein synthesis. Protein degradation half-times for actin variants were then determined by immunoblotting and fitting data to an exponential decay function.
DNase I Pulldown
DNase I conjugated Sepharose 4B beads were prepared and used to affinity purify actin from cell lysates, enabling the isolation of specific protein complexes.
Ribosome Isolation
Ribosomes were isolated from cell lysates via ultracentrifugation. Both supernatant and pellet fractions were subsequently collected and analyzed for protein content.
Polysome Gradient Analysis
Cell lysates were fractionated on sucrose density gradients to analyze polysome profiles. Proteins from the various fractions were then analyzed by immunoblotting.
Solubility Assay
Cell lysates were centrifuged to separate soluble and insoluble protein fractions. These fractions were then analyzed by immunoblotting to assess protein aggregation and solubility.
Single-Molecule Tracking in Live Cells
Two-color simultaneous imaging was performed on a Zeiss Elyra PS.1 super-resolution microscope in TIRF mode. Cells were sparsely labeled with Janelia Fluor HaloTag ligands and SNAP-tag ligands to enable precise visualization of individual molecules.
Two-color simultaneous imaging was performed on a Zeiss Elyra PS.1 super-resolution microscope in TIRF mode.
Individual particle tracking was conducted using the TrackMate plugin in Fiji, followed by sophisticated colocalization analysis with KNIME. Interactions were strictly defined by persistent colocalization for over 500 ms. Mean square displacement was utilized to determine diffusion coefficients. Statistical analysis of lifetimes and interaction events employed Welch’s ANOVA, Mann–Whitney U-test, and one-way ANOVA with Tukey’s post hoc test. Kinetic modeling, employing single or two-component exponential decay functions, was used to resolve distinct kinetic components of interaction lifetimes. The fraction of fluorescently labeled molecules was estimated via fluorescence intensity measurements and in-gel analysis.
Mass Spectrometry
For comprehensive total proteome analysis, cell pellets were prepared using SDC buffer, followed by ultrasonication, Lys-C and trypsin digestion, and subsequent desalting.
LC–MS/MS was performed using an Easy-nLC 1200 coupled with a QExactive HF mass spectrometer.
Liquid chromatography–mass spectrometry (LC–MS/MS) was performed using an Easy-nLC 1200 coupled with a QExactive HF mass spectrometer. Data were processed with MaxQuant, searching against the human proteome database. Label-free quantification (LFQ) and iBAQ values were calculated for robust protein quantification.
Selective Ribosome Profiling
Ribosome fractions were meticulously prepared following CHX treatment, DSP crosslinking, and RNase I digestion, with ribosomes subsequently isolated via sucrose cushions.
HaloTag pulldown was used to enrich specific ribosome–nascent chain complexes.
HaloTag pulldown was employed to enrich specific ribosome–nascent chain complexes. Ribosome-protected fragments (RPFs) were recovered, and libraries were prepared and sequenced. Data analysis involved trimming reads, extracting UMIs, mapping against non-coding RNA and the human genome, and calculating P-site offsets. Co-translational interactions of TRiC and PFD were identified through positional enrichment calculations using a two-tailed Fisher’s exact test with Benjamini-Hochberg correction.