Recombinant Xenopus laevis Anaphase-promoting complex subunit 16 (anapc16)

What is the function of ANAPC16 in Xenopus laevis?

ANAPC16 is a conserved subunit of the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase that plays a crucial role in cell cycle regulation . In Xenopus laevis, as in other metazoans, ANAPC16 is essential for proper APC/C activity toward mitotic substrates, facilitating timely chromosome segregation and mitotic exit . The protein is present in APC/C complexes throughout the cell cycle, suggesting a constitutive rather than cell-cycle-specific role . Unlike some other APC/C subunits, ANAPC16 appears to be important for APC/C function but not necessarily for the assembly of the APC/C holocomplex, as depletion studies have shown that other APC/C subunits can still form complexes in the absence of ANAPC16 .

Why is Xenopus laevis a suitable model organism for studying ANAPC16?

Xenopus laevis serves as an excellent model organism for studying ANAPC16 due to several advantageous characteristics. As an amphibian with phylogenetic positioning intermediate between aquatic vertebrates and land tetrapods, it provides valuable evolutionary context for understanding conserved cellular mechanisms . The Xenopus system offers practical advantages for laboratory research, including the ability to induce breeding through hormone injection and the availability of genetically-defined inbred strains and clones . Additionally, the evolutionary distance between Xenopus and mammals allows researchers to distinguish between species-specific adaptations and more fundamentally conserved features of cellular processes like cell cycle regulation . The University of Rochester maintains comprehensive resources for Xenopus laevis research, including transgenic animals, monoclonal antibodies, cell lines, and molecular probes that facilitate studies of proteins like ANAPC16 .

How conserved is ANAPC16 across different species?

ANAPC16 exhibits interesting evolutionary conservation patterns that provide insights into its function. Sequence homologs of ANAPC16 can be identified across metazoan species but appear to be absent in fungi . This suggests a metazoan-specific evolution or adaptation of this particular APC/C component. The conservation of ANAPC16 can be recognized through four primary sequence stretches that maintain similarity across species . Functional studies have provided evidence that the C. elegans gene K10D2.4 and the Danio rerio (zebrafish) gene zgc:110659 are functional equivalents of human APC16, demonstrating conservation of function across evolutionarily distant metazoan species . This pattern of conservation raises intriguing questions about why metazoan APC/C differs molecularly from unicellular APC/C and what specific selective pressures or functional requirements might have driven these differences .

What are the recommended methods for recombinant expression of Xenopus laevis ANAPC16?

For recombinant expression of Xenopus laevis ANAPC16, researchers should consider several methodological approaches based on established protocols for similar proteins. The ANAPC16 cDNA can be cloned into expression vectors such as pcDNA3.1 with appropriate tags (e.g., C-terminal DYKDDDDK tags) to facilitate purification and detection . When designing expression constructs, it's crucial to consider the relatively small size of ANAPC16 (approximately 12 kDa in humans) and optimize codon usage for the expression system of choice .

How can researchers effectively verify the association of ANAPC16 with the APC/C complex in Xenopus laevis?

Verification of ANAPC16 association with the APC/C complex in Xenopus laevis can be accomplished through multiple complementary approaches. Co-immunoprecipitation experiments represent a primary method, using antibodies against established APC/C subunits such as APC3/Cdc27 (part of the "head" domain) and APC4 (part of the "platform" domain) to pull down the complex and then detecting ANAPC16 by western blot . Reciprocal immunoprecipitations using anti-ANAPC16 antibodies should co-precipitate other APC/C subunits if ANAPC16 is genuinely part of the complex .

Size exclusion chromatography (gel filtration) provides another valuable approach. If ANAPC16 is a bona fide component of the APC/C, it should co-migrate with the APC/C holocomplex at approximately 1.5 MDa . Additionally, researchers can employ tandem affinity purification using tagged versions of ANAPC16 or other APC/C subunits, followed by mass spectrometry analysis to identify interacting proteins . For in vivo verification, fluorescently tagged ANAPC16 can be used to visualize co-localization with other APC/C components throughout the cell cycle in Xenopus cells or embryos, complementing the biochemical approaches described above.

What functional assays can determine the role of ANAPC16 in APC/C activity in Xenopus systems?

Several functional assays can effectively assess the role of ANAPC16 in APC/C activity within Xenopus systems. Cell cycle progression analysis using Xenopus egg extracts provides a powerful system to examine APC/C function. Researchers can immunodeplete endogenous ANAPC16 from the extracts and assess the impact on cell cycle progression, particularly at the metaphase-to-anaphase transition, by monitoring the degradation kinetics of known APC/C substrates such as cyclin B and securin .

Ubiquitination assays represent another critical approach. In vitro reconstitution of the ubiquitination activity using purified components (E1, E2, APC/C with or without ANAPC16, and substrate proteins) can directly measure the contribution of ANAPC16 to APC/C catalytic function . For in vivo functional assessment, morpholino-mediated knockdown in Xenopus embryos can be employed, followed by analysis of mitotic progression using phospho-histone H3 staining, similar to approaches used in zebrafish studies .

Additionally, researchers can perform rescue experiments by depleting endogenous ANAPC16 and introducing recombinant wild-type or mutant versions to identify functional domains. This approach has proven successful in human cells, where expression of EYFP-tagged APC16 reduced the prolonged mitosis phenotype caused by depletion of endogenous APC16 .

How does ANAPC16 contribute to APC/C structural integrity versus enzymatic activity?

The relationship between ANAPC16 and APC/C structural integrity versus enzymatic activity represents a nuanced aspect of APC/C biology. Research evidence indicates that ANAPC16 plays a more critical role in APC/C enzymatic function than in maintaining structural integrity . Studies of APC16-depleted cells revealed that the APC/C remained largely intact in terms of assembly and incorporation of other subunits (including APC3, 4, 5, 6, 8, and 13) despite the absence of ANAPC16 . This was demonstrated through gel filtration experiments showing that APC/C in both mock-depleted and ANAPC16-depleted cells had comparable size and contained key structural components .

What are the experimental considerations when investigating ANAPC16 phosphorylation states and their functional implications?

Investigating ANAPC16 phosphorylation states requires careful experimental design and technical considerations. Researchers should first determine baseline phosphorylation states throughout the cell cycle using Xenopus egg extracts synchronized at different cell cycle stages (interphase, prophase, metaphase, anaphase) . Phosphorylation can be detected through phospho-specific antibodies, if available, or by combining immunoprecipitation with mass spectrometry-based phosphopeptide analysis.

For functional studies, researchers should generate phosphomimetic (e.g., serine/threonine to glutamate) and phospho-deficient (serine/threonine to alanine) mutants at identified or predicted phosphorylation sites. These mutants can then be tested for their ability to rescue ANAPC16 depletion phenotypes in Xenopus systems . Additionally, identification of the kinases and phosphatases regulating ANAPC16 phosphorylation is crucial. Candidate kinases can be screened using in vitro kinase assays with purified recombinant ANAPC16, followed by in vivo validation through specific kinase inhibition or depletion.

How can researchers differentiate between direct and indirect effects when studying ANAPC16 depletion phenotypes?

Differentiating between direct and indirect effects of ANAPC16 depletion requires rigorous experimental approaches to establish causality. One essential strategy involves employing multiple independent depletion methods (e.g., RNAi, morpholinos, CRISPR/Cas9) targeting different regions of the ANAPC16 gene or transcript to confirm consistency of observed phenotypes . This helps rule out off-target effects specific to any single approach.

Rescue experiments provide crucial evidence for direct causality. Researchers should express RNAi-resistant versions of ANAPC16 (wild-type or with specific mutations) in depleted cells/embryos to determine which phenotypes can be rescued . The degree of rescue can be quantified using metrics such as the percentage of cells showing prolonged mitosis, as demonstrated in human cell studies where EYFP-APC16 expression reduced the fraction of cells with extended mitotic duration from 36% to 12% .

Time-resolved analysis is another valuable approach. By establishing the temporal sequence of cellular events following ANAPC16 depletion, researchers can distinguish primary effects (occurring immediately) from secondary consequences that develop later. Additionally, domain-specific mutations or truncations of ANAPC16 can help map specific functions to particular regions of the protein, providing mechanistic insights into how ANAPC16 contributes to APC/C activity .

For comprehensive analysis, researchers should examine both APC/C-dependent processes (substrate degradation, mitotic progression) and potentially APC/C-independent functions through techniques such as BioID or proximity labeling to identify the complete interactome of ANAPC16 beyond the core APC/C complex.

How do functional studies of ANAPC16 in Xenopus laevis compare with findings in other vertebrate models?

Comparative functional studies of ANAPC16 across vertebrate models reveal important evolutionary conservation and potential species-specific adaptations. In human cells, APC16 has been established as a bona fide subunit of the APC/C complex, present throughout the cell cycle and essential for proper APC/C function and mitotic progression . Studies in zebrafish (Danio rerio) demonstrated that morpholino-mediated knockdown of the ANAPC16 homolog zgc:110659 resulted in increased phospho-histone H3 staining in the head region of developing embryos, indicating mitotic arrest similar to that observed upon depletion of other APC/C subunits like APC11 .

This phenotypic consistency across evolutionary distant vertebrates suggests fundamental conservation of ANAPC16 function in APC/C activity. When comparing human and Xenopus laevis ANAPC16, researchers should note the four conserved primary sequence stretches that define this protein family across metazoans . These conserved regions likely represent functional domains critical for interaction with other APC/C subunits or for influencing APC/C catalytic activity.

Interestingly, while ANAPC16 is conserved among metazoans, it appears to be absent in fungi, raising questions about how unicellular organisms achieve comparable APC/C functionality without this subunit . This evolutionary divergence provides an opportunity to investigate how metazoan APC/C has adapted to potentially more complex regulatory requirements in multicellular organisms. Comparative studies across Xenopus, human, and zebrafish systems can help identify both conserved core functions and potential species-specific adaptations of ANAPC16 in vertebrate cell cycle regulation.

What can researchers learn from the absence of ANAPC16 homologs in fungi despite conservation across metazoan species?

The absence of identifiable ANAPC16 homologs in fungi despite conservation across metazoan species represents an intriguing evolutionary puzzle with significant implications for understanding APC/C function and adaptation . This pattern suggests that ANAPC16 emerged as an APC/C component after the divergence of metazoans from fungi, potentially as an adaptation to multicellularity or more complex developmental programs.

Researchers can leverage this evolutionary distinction to investigate several key questions. First, comparative biochemical studies between fungal and metazoan APC/C can reveal how fungal APC/C achieves functional competence without ANAPC16. This may involve compensatory functions distributed among other APC/C subunits or unique regulatory mechanisms. Second, researchers can explore whether ANAPC16 provides metazoan APC/C with enhanced substrate specificity, regulatory control, or adaptation to tissue-specific requirements absent in unicellular organisms.

Structural biology approaches comparing APC/C architecture between fungi and metazoans could identify conformational differences that might explain functional distinctions. Additionally, evolutionary analyses of ANAPC16 sequence conservation across metazoan lineages may identify signatures of selection that correlate with increased organismal complexity or specific developmental innovations.

The APC/C serves as the principal target of the mitotic checkpoint, which prevents chromosome segregation while chromosomes remain unattached to spindle microtubules . Researchers should investigate whether ANAPC16 plays a role in metazoan-specific aspects of this checkpoint regulation, potentially allowing for more nuanced control of mitotic progression in the context of complex multicellular development.

What are common challenges in purifying functional recombinant Xenopus laevis ANAPC16 and how can they be addressed?

Purification of functional recombinant Xenopus laevis ANAPC16 presents several challenges that require specific optimization strategies. One common issue is the relatively small size of ANAPC16 (approximately 12 kDa), which can complicate expression and purification . Researchers can address this by using fusion tags that increase protein size and stability, such as GST, MBP, or SUMO tags, with appropriate protease cleavage sites for tag removal if necessary. Selection of the expression system is also critical, with E. coli being suitable for structural studies but potentially lacking proper folding or post-translational modifications, while insect or mammalian cells may better preserve native conformation.

Protein solubility challenges can be addressed through optimization of buffer conditions during purification. Testing various pH ranges (typically 6.5-8.5), salt concentrations (100-500 mM NaCl), and addition of stabilizing agents (glycerol 5-10%, reducing agents like DTT or BME) can significantly improve solubility and stability. For proteins prone to aggregation, arginine and glutamate additives (50-100 mM) have proven effective in enhancing solubility without interfering with function.

To ensure functional activity of purified ANAPC16, researchers should verify proper folding using circular dichroism spectroscopy and assess APC/C incorporation capability through in vitro reconstitution assays with other purified APC/C subunits . Co-expression with interacting APC/C subunits may also enhance solubility and native folding. Finally, storage conditions require optimization, with flash-freezing in small aliquots typically recommended to maintain functional integrity through multiple freeze-thaw cycles.

What control experiments are essential when studying the effects of ANAPC16 depletion in Xenopus systems?

When studying ANAPC16 depletion effects in Xenopus systems, several essential control experiments must be included to ensure reliable and interpretable results. First, researchers should verify depletion efficiency using both Western blot analysis and, where possible, immunofluorescence to confirm protein reduction at both population and single-cell levels . Multiple independent depletion methods (different siRNAs, morpholinos targeting different regions) should be employed to rule out off-target effects, with consistent phenotypes across different targeting strategies providing stronger evidence for specificity .

Rescue experiments constitute a critical control. Expression of RNAi-resistant wild-type ANAPC16 should reverse depletion phenotypes, as demonstrated in human cell studies where expression of EYFP-APC16 reduced the fraction of cells with prolonged mitosis from 36% to 12% . To control for general effects of experimental manipulation, untransfected cells from the same preparations should be analyzed in parallel, which should show consistent depletion phenotypes regardless of the treatment of transfected populations .

Additionally, researchers should compare ANAPC16 depletion phenotypes with those resulting from depletion of other APC/C subunits to distinguish ANAPC16-specific effects from general APC/C dysfunction . Temporal controls are also important; analysis at multiple time points after depletion can distinguish primary from secondary effects. Finally, functional readouts should include multiple metrics of APC/C activity, such as substrate degradation kinetics, mitotic duration, and chromosome segregation accuracy, to comprehensively characterize the consequences of ANAPC16 depletion.

How can researchers optimize co-expression systems for studying ANAPC16 interactions with other APC/C subunits?

Optimizing co-expression systems for studying ANAPC16 interactions with other APC/C subunits requires careful consideration of expression vectors, host systems, and purification strategies. For vector design, researchers should use compatible vectors with different antibiotic selection markers and controllable promoters (e.g., pET, pACYC, pCDF for bacterial systems; or dual-promoter vectors for mammalian/insect cells). Differential tagging is crucial—placing distinct affinity tags (His, FLAG, Strep, etc.) on different subunits enables sequential affinity purification to isolate specific complexes .

The choice of expression system is critical. While E. coli is simpler and higher-yielding, insect cell systems (particularly baculovirus-infected Sf9 or Hi5 cells) better support the expression of functional multi-subunit complexes like APC/C. For complex reconstitution studies, researchers can employ the MultiBac system, which allows integration of multiple genes into a single baculovirus for synchronized expression of numerous subunits.

For verifying interactions, initial screening can be performed using split-reporter systems (e.g., yeast two-hybrid, split-luciferase) to identify direct interaction partners of ANAPC16 within the APC/C complex. Follow-up confirmation through co-immunoprecipitation and label-transfer approaches provides more detailed interaction information . When purifying complexes, tandem affinity purification using tags on different subunits effectively isolates specific subcomplexes, while size exclusion chromatography as a final step separates fully assembled complexes from partial assemblies or excess subunits .

To assess the functional significance of interactions, researchers should design mutational studies targeting predicted interaction interfaces, with comprehensive alanine-scanning or domain deletion approaches helping to map critical interaction regions. Finally, structural characterization using techniques like cryo-EM or crosslinking mass spectrometry can provide detailed spatial arrangement information for ANAPC16 within the assembled APC/C complex.