Recombinant Candida glabrata 60S ribosomal protein L43 (RPL43)

What is the structural location and functional significance of RPL43 in C. glabrata ribosomes?

RPL43 is strategically located near the E-site of ribosomes and in close proximity to the peptidyl transferase center. This positioning is critical for its function in ribosome assembly and stability. The protein contains highly positive charges and disordered extensions that insert into ribosomes to facilitate interactions with rRNAs and other ribosomal proteins . These structural features enable RPL43 to coordinate conformational changes within the ribosomal complex, particularly during the production of 7S rRNA, which is essential for mature 60S ribosomal subunit formation .

How many homologs of RPL43 exist in C. glabrata and what is their relationship?

Based on research in related yeast species, C. glabrata contains two homologs of RPL43: RPL43A and RPL43B. These homologs share approximately 90% identity at the DNA sequence level but maintain 100% identity in amino acid sequence . This suggests functional redundancy despite different transcriptional regulation. While deletion of either RPL43A or RPL43B individually results in only minor growth defects, simultaneous deletion of both homologs is lethal, confirming the essential nature of this ribosomal protein .

What is the temporal incorporation pattern of RPL43 during ribosome biogenesis?

RPL43 is incorporated into pre-ribosomal particles as early as the 90S pre-ribosome stage, although its major integration occurs during the nucleolar phase of pre-60S formation . This temporal incorporation pattern has been established through immunoprecipitation experiments using transacting factors at different stages of ribosome assembly. The proper incorporation of RPL43 is crucial for subsequent pre-rRNA processing steps, particularly the late stages of 7S rRNA processing .

Which proteins interact with RPL43 during its incorporation into ribosomes?

RPL43 forms a trimeric complex with the proteins Puf6 and Loc1, which function as dedicated chaperones for this ribosomal protein . These interactions are critical for maintaining RPL43 stability and facilitating its proper incorporation into pre-60S ribosomal particles. The association occurs co-translationally via the N-terminus of RPL43 . The N-terminus of Puf6 contains nuclear localization signals for transport, while its PUF (Pumilio) domain is essential for interaction with Loc1, RPL43, and 60S subunits. The C-terminus of Loc1 is particularly important for interaction with both Puf6 and RPL43 .

How do chaperone proteins regulate RPL43 stability and what experimental evidence supports this?

Puf6 and Loc1 are crucial for maintaining RPL43 protein stability. Experimental evidence demonstrates that:

• In puf6Δ strains, RPL43 protein levels decrease slightly

• In loc1Δ strains, RPL43 protein levels decrease significantly

• Overexpression of both Puf6 and Loc1 together increases RPL43 levels approximately 2.1-fold

These findings were established through western blot analysis of RPL43 levels under various genetic conditions. Additionally, cycloheximide chase experiments showed that the stability of newly synthesized RPL43 decreased slightly in puf6Δ and significantly in loc1Δ strains . This evidence collectively indicates that Puf6 and Loc1 function as dedicated chaperones that protect nascent RPL43 from degradation.

What are the consequences of RPL43 depletion on ribosome assembly?

Depletion of RPL43 has significant consequences for ribosome assembly. When RPL43 expression is repressed (using a GAL-driven promoter system in glucose media), cells exhibit characteristic pre-rRNA processing defects, including:

• Accumulation of 35S pre-rRNA

• Accumulation of 27S, 27SA2, and 23S pre-rRNA species

• Blockage of C2 cleavage in the pre-rRNA processing pathway

These processing defects closely resemble those observed in puf6Δ and loc1Δ mutants, highlighting the functional relationship between these proteins . Additionally, while RPL43 depletion does not affect the loading of Puf6 and Loc1 onto pre-60S particles, it impairs their subsequent release, indicating that RPL43 incorporation is required for the release of these chaperones during ribosome maturation .

What expression systems are optimal for producing recombinant C. glabrata RPL43?

For optimal expression of recombinant C. glabrata RPL43, researchers should consider the following approaches:

For all systems, maintaining buffer conditions that stabilize RPL43 is critical, including the addition of RNase inhibitors, appropriate salt concentration (300-500 mM NaCl), and reducing agents. Co-expression with chaperones Puf6 and Loc1 may significantly improve stability and solubility of the recombinant protein.

How can researchers analyze RPL43 incorporation into pre-ribosomal particles?

To study RPL43 incorporation into pre-ribosomal particles, several complementary techniques can be employed:

• Immunoprecipitation of Pre-ribosomal Complexes:

  • Tag RPL43 with epitope tags (HA, FLAG, or TAP)

  • Precipitate complexes using tag-specific antibodies

  • Analyze co-precipitating pre-rRNAs by northern blotting with specific probes

  • Identify associated proteins by mass spectrometry

• Gradient Centrifugation Analysis:

  • Separate complexes on 10-50% sucrose gradients

  • Analyze fractions for the presence of RPL43 by western blotting

  • Correlate with pre-rRNA species by northern blotting with probes targeting specific regions (as shown in Figure 1B in the reference)

• RNA Processing Analysis:

  • Use specific probes to detect intermediate rRNA species (35S, 27S, 27SA2, 23S)

  • Compare processing patterns between wild-type and mutant strains

  • Quantify the accumulation of precursor rRNAs relative to mature rRNAs

What approaches can be used to study the stability of nascent RPL43?

To study the stability of nascent RPL43, researchers can implement the following methodological approaches:

• Cycloheximide Chase Assays:

  • Add cycloheximide to inhibit protein synthesis

  • Collect samples at various time points (0-60 minutes)

  • Separate free and ribosome-incorporated RPL43 by ultracentrifugation

  • Analyze protein levels by western blotting

• Pulse-Chase Experiments:

  • Label newly synthesized proteins with radioisotopes or non-radioactive labels

  • Chase with unlabeled amino acids

  • Immunoprecipitate RPL43 at different time points

  • Quantify labeled protein decay over time

• Fractionation Analysis:

  • Separate cellular compartments (cytosol, nucleus, nucleolus)

  • Track RPL43 localization and stability in different compartments

  • Compare wild-type with chaperone deletion strains (puf6Δ, loc1Δ)

What is known about RPL43 sequence variations in clinical C. glabrata isolates?

Analysis of genomic variation across clinical C. glabrata isolates can provide insights into potential roles of RPL43 in pathogenesis and drug resistance:

• Genomic Variation Analysis:

  • Whole genome sequencing of clinical isolates has revealed extensive SNPs and INDELs across C. glabrata strains

  • While specific data on RPL43 variations is not directly provided in the search results, the approach used for analyzing drug resistance genes can be applied to RPL43

• Mutation Pattern Assessment:

  • Similar to other genes in C. glabrata, missense mutations represent approximately 83-89% of variants, followed by frameshift mutations

  • Analysis should focus on whether RPL43 mutations correlate with specific phenotypes like drug resistance or virulence

• Functional Impact Prediction:

  • Any identified variants would need experimental validation to determine if they are gain-of-function mutations or mutations that impair protein function

  • Critical analysis should distinguish between natural population diversity and clinically significant variations

How might RPL43 function intersect with DNA damage response pathways in C. glabrata?

The intersection of ribosome biogenesis and DNA damage response represents an intriguing area for future research:

• Potential Regulatory Connections:

  • C. glabrata shows a unique response to DNA alkylating agents like methyl methanesulfonate (MMS), with homologous recombination (HR) playing a key role in repair

  • Similar to histone H4 downregulation during MMS stress , RPL43 levels might be modulated during DNA damage response

• Stress-Specific Translation Regulation:

  • During DNA damage, specific translation patterns may be required for repair proteins

  • RPL43's role in ribosome biogenesis could influence the translation of DNA repair factors

• Shared Regulatory Factors:

  • Proteins like CgCmr1 (identified as interacting with histone H4) might also influence ribosomal protein regulation

  • Investigating potential shared regulators between chromatin components and ribosomal proteins could reveal novel stress response mechanisms

What experimental approaches could elucidate the role of RPL43 in translational regulation during stress?

To investigate RPL43's potential role in stress-specific translational regulation:

• Ribosome Profiling Under Stress Conditions:

  • Apply ribosome profiling (Ribo-seq) to wild-type and RPL43-depleted cells under various stresses

  • Identify differentially translated mRNAs

  • Map changes in translation efficiency to specific stress response pathways

• Selective Ribosome Profiling:

  • Tag RPL43 to isolate specific ribosome populations

  • Determine if RPL43-containing ribosomes preferentially translate specific mRNA subsets

  • Compare normal versus stress conditions to identify specialized translational functions

• Integrated Multi-omics Approach:

  • Combine transcriptomics, proteomics, and ribosome profiling

  • Correlate changes in RPL43 levels with alterations in gene expression and protein synthesis

  • Develop network models of RPL43's role in stress-specific translation