Recombinant Mouse Nuclear pore complex protein Nup54 (Nup54)

What is Nup54, and what role does it play in the nuclear pore complex?

Nup54 is a nucleoporin protein that forms part of the central channel of the nuclear pore complex (NPC). The NPC is a large macromolecular assembly embedded in the nuclear envelope, facilitating selective nucleocytoplasmic transport of macromolecules such as RNA and proteins. Nup54, along with its molecular partners Nup58 and Nup62, constitutes the flexible scaffold of the NPC's transport channel. Structurally, Nup54 contributes to the ring-like architecture of the channel, enabling dynamic rearrangements that regulate transport activity .

Functionally, Nup54 is involved in maintaining genome integrity. It has been shown to play a role in homologous recombination (HR)-mediated DNA repair processes, particularly in response to DNA double-strand breaks (DSBs). Depletion of Nup54 increases cellular sensitivity to ionizing radiation (IR), leading to mitotic catastrophe and chromosomal aberrations . These findings underscore its importance not only in nucleocytoplasmic transport but also in DNA damage response mechanisms.

How is recombinant mouse Nup54 produced for research purposes?

Recombinant mouse Nup54 is typically produced using molecular cloning techniques. The gene encoding Nup54 is cloned into an expression vector, which is then introduced into a suitable host system such as Escherichia coli or mammalian cells. The expressed protein can be purified using affinity chromatography methods, such as nickel-nitrilotriacetic acid (Ni-NTA) chromatography if a His-tag is included in the construct.

For functional studies, researchers often use constructs that allow for post-translational modifications or specific tagging (e.g., HA-tag or GFP-tag). These modifications enable downstream applications such as immunoprecipitation, fluorescence microscopy, or interaction studies .

What experimental systems are commonly used to study Nup54 function?

Several experimental systems are employed to investigate Nup54's function:

• Cellular Models: Knockdown or knockout studies using siRNA or CRISPR/Cas9 technologies help elucidate its role in DNA repair and NPC assembly.

• Biochemical Assays: Interaction studies with other nucleoporins like Nup62 and Nup58 are conducted using co-immunoprecipitation or yeast two-hybrid assays.

• Structural Studies: Crystallography and cryo-electron microscopy are used to determine the molecular architecture of Nup54 within the NPC .

• In Vivo Models: Transgenic mouse models expressing mutant forms of Nup54 provide insights into its physiological roles.

What structural features of Nup54 are critical for its function?

Nup54 contains phenylalanine-glycine (FG) repeat regions that interact with transport receptors during nucleocytoplasmic transport. These FG repeats are essential for selective permeability within the NPC. Additionally, structural studies have revealed that Nup54 forms triple-helical interactions with Nup62 and contributes to a flexible ring structure with Nup58. This flexibility allows for diameter changes in the NPC channel, accommodating various cargo sizes .

Mutations affecting these structural features can disrupt NPC function and lead to defects in transport activity or genome integrity maintenance .

How does Nup54 contribute to homologous recombination repair?

Nup54 plays a pivotal role in homologous recombination (HR)-mediated DNA repair by facilitating the efficient repair of DNA double-strand breaks (DSBs). Depletion of Nup54 has been shown to reduce HR repair activity significantly. This reduction correlates with decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges following exposure to ionizing radiation .

Mechanistically, Nup54 appears to interact epistatically with Rad51, a key protein in HR repair pathways. Loss of Nup54 disrupts Rad51-mediated strand invasion processes necessary for accurate DSB repair . Further studies suggest that this role may involve the recruitment of damaged DNA regions to nuclear pore complexes for efficient repair.

What are the implications of Nup54 depletion on cell cycle progression?

Depletion of Nup54 has profound effects on cell cycle progression:

• G2/M Arrest: Cells deficient in Nup54 exhibit prolonged G2 arrest following DNA damage induced by ionizing radiation. This delay allows cells additional time to attempt DNA repair but ultimately leads to mitotic catastrophe if repair fails.

• Chromosomal Aberrations: Increased formation of chromosomal aberrations is observed in cells lacking functional Nup54. These aberrations arise from unresolved DNA damage during replication .

These findings highlight the dual role of Nup54 in cell cycle regulation and genome stability.

How does structural flexibility in the NPC channel influence transport activity?

The structural flexibility of the NPC channel, mediated by proteins like Nup54, is critical for regulating transport activity. The central channel undergoes dynamic rearrangements facilitated by conformational changes in nucleoporins such as Nup58 and Nup62. These rearrangements alter the channel diameter from approximately 20 nm to 40 nm, accommodating cargoes of varying sizes .

Nup54 contributes to this flexibility through its interaction with other nucleoporins and FG repeat regions that bind transport receptors. Disruptions in these interactions can impair selective permeability and lead to transport defects.

Are there any known post-translational modifications (PTMs) of mouse Nup54?

Post-translational modifications (PTMs) such as phosphorylation, ubiquitination, and SUMOylation have been implicated in regulating nucleoporin functions, including those of Nup54. For instance:

• Ubiquitination: Studies suggest that ubiquitination-dependent binding may be involved in DNA damage response pathways linked to NPCs.

• SUMOylation: SUMOylation pathways mediate interactions between persistent DSBs and NPCs for efficient repair .

Further research is needed to map specific PTM sites on mouse Nup54 and their functional implications.