Recombinant Zygosaccharomyces rouxii Nuclear rim protein 1 (NUR1)

What is the basic structure of Zygosaccharomyces rouxii Nuclear Rim Protein 1?

Z. rouxii Nuclear Rim Protein 1 (NUR1) is a protein localized to the nuclear rim, with a full length of 511 amino acids. The protein contains several key structural elements, with the N-terminal region (amino acids 1-103) being particularly significant for its localization to the nuclear envelope. The N-terminal portion contains an amphipathic helix within the first 20 amino acids that serves as an inner peripheral membrane (IPM) anchor. The protein sequence includes hydrophobic residues (Leu2, Val5, Trp9, Phe12, Phe13) that are critical for membrane targeting. Beyond the N-terminal region, NUR1 also contains a central coiled-coil domain (approximately residues 174-219) and a C-terminal domain (residues 220-319), though the N-terminal region appears to be sufficient for essential functions at lower temperatures .

How does the N-terminal region of NUR1 contribute to its localization and function?

The N-terminal region of NUR1, particularly amino acids 1-103, is sufficient for targeting the protein to the nuclear envelope. Within this region, residues 1-20 contain an amphipathic helix that functions as an inner peripheral membrane (IPM) anchor. Experimental evidence shows that fusion of just the first 20 amino acids of NUR1 to a reporter protein (cNLS-GFP) is sufficient to target the fusion protein to the nuclear rim. Mutations that disrupt the hydrophobic face of this helix, such as replacing hydrophobic residues (Leu2, Val5, Trp9, Phe12, Phe13) with alanine, abolish membrane localization and essential function. This indicates that the N-terminal IPM anchor is the major membrane localization signal within NUR1 and is critical for its function .

What is known about NUR1's interactions with membrane lipids?

Research indicates that NUR1's interaction with membranes is direct rather than mediated through other membrane proteins. Liposome-binding assays have shown that NUR1 can directly interact with phosphatidylcholine liposomes, dependent on its IPM motif. When the IPM anchor is inactivated either through deletion of the motif (residues 1-14) or through point mutations (5A mutant), the protein loses its ability to interact with membranes. This direct lipid interaction is critical for NUR1's localization to the inner nuclear membrane and for its essential cellular functions. The specificity of this interaction may be determined by the physicochemical properties of the amphipathic helix within the IPM anchor .

What are the optimal conditions for storing and handling recombinant NUR1 protein?

Recombinant Z. rouxii NUR1 protein requires specific storage conditions to maintain stability and functionality. The protein should be stored at -20°C to -80°C, with the shelf life of liquid formulations typically being 6 months and lyophilized formulations lasting up to 12 months. For working aliquots, storage at 4°C for up to one week is recommended. Repeated freezing and thawing should be avoided as this can lead to protein degradation. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and addition of glycerol to a final concentration of 5-50% is recommended for long-term storage. Before opening, vials should be briefly centrifuged to bring contents to the bottom .

How can researchers effectively visualize NUR1 localization in cellular systems?

Visualization of NUR1 localization can be effectively achieved through fluorescence microscopy using GFP fusion constructs. Studies have successfully used various NUR1-GFP fusion proteins to determine its subcellular localization. For optimal results, researchers should consider creating fusion constructs with different portions of the NUR1 protein (e.g., full-length, N-terminal region 1-103, or specific domains) to determine which regions are necessary and sufficient for proper localization. Complementary approaches include immuno-electron microscopy, which has revealed that NUR1 localizes specifically to the inner nuclear membrane rather than being distributed throughout the nuclear envelope. When overexpressed, NUR1 constructs containing the N-terminal region can induce formation of intranuclear membranes, which should be taken into consideration when interpreting localization data .

What strategies can be used to determine the functional domains of NUR1?

To determine the functional domains of NUR1, researchers have successfully employed several complementary approaches:

• Truncation analysis: Creating a series of truncated constructs (e.g., NUR1-(1-173), NUR1-(174-319)) and testing their ability to provide essential functions in cells lacking endogenous NUR1.

• Site-directed mutagenesis: Introducing specific mutations in key regions, such as the 5A mutant where hydrophobic residues in the N-terminal IPM anchor are replaced with alanine.

• Domain fusion experiments: Fusing specific domains of NUR1 to reporter proteins like GFP to assess their localization properties.

• Functional complementation assays: Testing whether different constructs can restore growth in cells where endogenous NUR1 has been depleted.

• Membrane interaction studies: Using liposome binding assays to determine regions required for membrane interaction.

These approaches have revealed that the N-terminal region (1-173) is sufficient for essential functions at lower temperatures, while the C-terminal portion (174-319) alone is not functional. Furthermore, the integrity of the IPM anchor within the N-terminal region is critical for both function and localization .

How can the NUR1 system be utilized for manipulation of large DNA regions in plant chromosomes?

The recombination system related to Z. rouxii, particularly the R gene of plasmid pSR1, has shown significant potential for DNA manipulation in plant systems. This system operates through site-specific recombination between specific recombination sites (RSs), leading to either excision or inversion of DNA segments flanked by these sites. Research has demonstrated efficient functioning of this system in tobacco cells, where the R protein catalyzes precise recombination between RS copies. This capability provides a foundation for developing DNA technology that can manipulate large regions of DNA in plant chromosomes.

Researchers can implement this system by:

• Designing constructs with RS sites flanking regions of interest

• Co-introducing the R gene to express the recombinase

• Using reporter systems (such as cryptic β-glucuronidase genes) to monitor recombination events

This approach has been successful in both transient expression systems (through electroporation of protoplasts) and with genes stably integrated into chromosomes. The system's precision and efficiency make it particularly valuable for complex genetic manipulations in plant biotechnology applications .

What are the implications of NUR1 overexpression on nuclear membrane structure?

Overexpression of NUR1, particularly constructs containing the N-terminal region (e.g., NUR1-(1-103)-GFP), has been observed to induce the formation of intranuclear membranes. Electron microscopy analysis has revealed that these structures form within the nucleus when the protein is overproduced. This phenomenon is not unique to NUR1 but has been observed with other proteins containing amphipathic helices, such as Nup53. The induction of these membranous structures suggests that NUR1 has the capacity to influence membrane organization and proliferation.

The implications of this phenomenon include:

• Potential disruption of normal nuclear architecture and function when NUR1 is overexpressed

• Insights into mechanisms of nuclear membrane biogenesis and organization

• Possible applications in engineering nuclear membrane structures for research purposes

Researchers working with NUR1 overexpression systems should be aware of these effects when interpreting experimental results, as the induced membrane structures may influence other nuclear processes .

How does NUR1 compare with related nuclear rim proteins from other species?

While the search results don't provide direct comparative information about NUR1 homologs across species, some insights can be drawn from the related Nbp1 protein mentioned in the research. Both appear to share mechanisms for targeting to the inner nuclear membrane through amphipathic helices that function as IPM anchors. This targeting mechanism, involving the combination of an N-terminal IPM anchor and a nuclear localization signal (NLS), appears to be a conserved strategy for inner nuclear membrane protein localization.

Researchers interested in comparative analysis should consider:

• Sequence alignment of NUR1 with potential homologs in other yeast species and higher eukaryotes

• Functional complementation experiments to determine if proteins from other species can substitute for Z. rouxii NUR1

• Structural analysis of the N-terminal amphipathic helices to identify conserved features that enable membrane targeting

Such comparative analyses could provide evolutionary insights into nuclear envelope organization and the specialized functions of nuclear rim proteins across different organisms .

What are common challenges in working with recombinant NUR1 and how can they be addressed?

Working with recombinant NUR1 presents several challenges that researchers should be prepared to address:

Additionally, when designing experiments with NUR1 constructs, researchers should be aware that the N-terminal region is critical for function and localization. Truncated or mutated constructs may behave differently than the full-length protein. When visualizing NUR1 localization, overexpression may induce formation of intranuclear membranes, which should be considered when interpreting results .

How can researchers distinguish between specific and non-specific effects when studying NUR1 function?

To distinguish between specific and non-specific effects when studying NUR1 function, researchers should implement several control strategies:

• Use multiple construct designs: Compare results from full-length NUR1, truncated variants, and point mutants to identify which effects are associated with specific domains.

• Include appropriate negative controls: Use proteins with similar biochemical properties but different functions, or mutated versions of NUR1 that lack specific functional domains.

• Perform dose-response experiments: Test a range of protein concentrations to identify potential threshold effects or artifacts from overexpression.

• Validate with complementary approaches: Combine in vitro assays (such as liposome binding) with in vivo localization studies and functional complementation to build a consistent picture of NUR1 activity.

• Conduct rescue experiments: If a phenotype is observed following NUR1 manipulation, attempt to rescue it with wild-type NUR1 to confirm specificity.

The research has demonstrated that specific effects of NUR1 are strongly linked to its N-terminal IPM anchor, as mutations in this region abolish both membrane localization and function. This provides a basis for distinguishing specific effects mediated through membrane interaction from potential non-specific effects .

What controls are essential when investigating NUR1 localization and membrane interactions?

When investigating NUR1 localization and membrane interactions, several essential controls should be included:

• Negative targeting controls: Constructs lacking the IPM anchor (such as NUR1-(15-319) or NUR1 with 5A mutations in the N-terminal helix) to demonstrate the specificity of membrane targeting.

• Nuclear localization controls: Constructs with only a nuclear localization signal (such as cNLS-GFP) to distinguish between general nuclear accumulation and specific enrichment at the nuclear envelope.

• Protein integrity verification: Western blot analysis to confirm that fusion proteins remain intact and are not degraded, which could lead to misinterpretation of localization data.

• Multiple visualization methods: Combining different approaches (such as fluorescence microscopy and electron microscopy) to confirm localization patterns.

• Membrane specificity controls: When using liposome binding assays, include liposomes of different compositions to test for specificity of membrane interactions.

• Functional correlation: Assess whether constructs that show altered localization also show corresponding changes in functional assays to establish the biological relevance of localization patterns.

Research has shown that while full-length NUR1 and the N-terminal region (1-103) localize to the nuclear rim, constructs with disrupted IPM anchors accumulate within the nucleus but are not enriched at the nuclear envelope, demonstrating the importance of these controls for accurate interpretation .

What are promising areas for future research on NUR1's role in nuclear organization?

Several promising directions for future research on NUR1's role in nuclear organization include:

• Interaction network mapping: Identifying proteins that interact with NUR1 at the nuclear rim to understand its role in larger complexes governing nuclear envelope structure and function.

• Dynamic regulation: Investigating how NUR1 localization and function might be regulated during different cell cycle stages or in response to cellular stresses.

• Chromatin interactions: Exploring potential roles of NUR1 in tethering chromatin to the nuclear periphery, which could influence gene expression and genome organization.

• Comparative genomics: Analyzing NUR1 homologs across fungal species to understand evolutionary conservation and divergence of nuclear rim proteins.

• Structural biology: Determining the three-dimensional structure of NUR1, particularly focused on how the amphipathic helix interacts with membrane lipids.

• Cell size regulation: Investigating the observation that cells lacking functional NUR1 IPM anchors are larger than wild-type cells, potentially indicating a role in ploidy regulation or cell cycle control.

These research directions could provide significant insights into fundamental aspects of nuclear envelope biology and chromosome organization .

How might advanced imaging techniques enhance our understanding of NUR1 dynamics?

Advanced imaging techniques could substantially enhance our understanding of NUR1 dynamics in several ways:

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED microscopy could reveal the precise distribution of NUR1 within the nuclear rim at nanometer resolution, potentially identifying specific microdomains or patterns not visible with conventional microscopy.

• Live-cell imaging: Tracking NUR1-fluorescent protein fusions in living cells could provide insights into the protein's dynamics during cell cycle progression, membrane reorganization during mitosis, or responses to cellular stresses.

• FRAP (Fluorescence Recovery After Photobleaching): This technique could measure the mobility of NUR1 within the inner nuclear membrane, providing information about whether it forms stable complexes or dynamically exchanges.

• Correlative light and electron microscopy (CLEM): Combining fluorescence and electron microscopy could connect NUR1 localization with ultrastructural features of the nuclear envelope.

• Single-molecule tracking: Following individual NUR1 molecules could reveal heterogeneity in behavior and identify different subpopulations with distinct dynamic properties.

• Advanced electron microscopy: Techniques such as cryo-electron tomography could provide structural insights into how NUR1 organizes nuclear membrane architecture, particularly in contexts where overexpression induces intranuclear membrane formation.

These approaches would extend beyond the static localization data currently available and provide a more dynamic understanding of NUR1's behavior in cellular contexts .