Recombinant Mouse RUN and FYVE domain-containing protein 1 (Rufy1)

What is the domain architecture of RUFY1 and how does it relate to its function?

RUFY1 belongs to the RUFY protein family, which includes four members in mammals: RUFY1, RUFY2, RUFY3, and RUFY4. As indicated by the name, RUFY1 contains a RUN domain and a FYVE domain . The RUN domain is located at the N-terminus and is critical for protein-protein interactions, particularly binding to small GTPases. The presence of conserved arginine residues (R206 and R208) within the RxRAWL motif of the RUN domain is essential for interaction with Arl8b, an Arf-like GTP-binding protein . The FYVE domain facilitates endosomal localization, while the C-terminal coiled-coil region mediates interaction with the dynein-dynactin complex, enabling dynein-dependent organelle clustering .

What RUFY1 isoforms have been identified in mouse models?

Two primary isoforms of RUFY1 have been identified: a longer isoform (~80 kD) and a shorter isoform (~70 kD). These isoforms differ in the first 108 amino acids at the N-terminus . Expression analysis has revealed that the relative abundance of these isoforms varies across cell types. For instance, in HeLa cells, the longer isoform (~80 kD) is more abundant than the shorter isoform (~70 kD), whereas HEK293T cells exhibit nearly equal expression of both isoforms .

What is the subcellular localization pattern of RUFY1?

RUFY1 predominantly localizes to a subset of endosomes that are positive for Rab14, with modest colocalization with early endosomal proteins like EEA1 and SNX1 . Importantly, RUFY1 does not colocalize with LAMP1 or Lysotracker dye, indicating absence from late endosomes and lysosomes . RUFY1-positive endosomes also contain CI-M6PR and partially overlap with Vps26-positive structures, suggesting involvement in retromer-mediated trafficking .

How does RUFY1 interact with Arl8b and what determines the specificity of this interaction?

RUFY1 binds specifically to the GTP-bound (active) form of Arl8b but not to the GDP-bound (inactive) form, as demonstrated by GST pulldown and co-immunoprecipitation assays . This interaction is mediated by the RUN domain of RUFY1, particularly through conserved arginine residues (R206 and R208) within the RxRAWL motif . Deletion of the RUN domain (RUFY1 ΔRUN) or mutation of these residues (RUFY1 R206A/R208A) drastically reduces Arl8b binding . Furthermore, direct protein-protein interaction assays using purified proteins have confirmed that the RUN domain fragment (1-302 amino acids) of RUFY1 preferentially binds to the wild-type and constitutively active (Q75L) forms of Arl8b compared to the inactive (T34N) form .

Which Rab proteins interact with RUFY1 and what is their functional significance?

RUFY1 interacts with multiple early endosomal Rab proteins, including Rab4, Rab5, and Rab14 . Among these, Rab14 appears to be particularly important as it is essential for RUFY1 localization to early/sorting endosomes . The interaction between RUFY1 and Rab14 is functionally significant as it contributes to the regulation of receptor recycling and cargo sorting from early endosomes .

How does RUFY1 compare to other RUFY family members in terms of Arl8b binding?

While all RUFY family members contain RUN domains, they exhibit differential binding to Arl8b. Recent studies have shown that Arl8b interacts with and regulates the lysosomal localization of RUFY3 and RUFY4 . Interestingly, quantitative binding assays have demonstrated that RUFY3 binds to Arl8b with approximately twofold higher affinity than RUFY1 . Additionally, the Arl8b-binding site in RUFY3 has been identified as the C-terminal coiled-coil region rather than the RUN domain, indicating distinct binding mechanisms within the RUFY family .

What is the role of RUFY1 in CI-M6PR trafficking and lysosomal cargo sorting?

RUFY1 plays a critical role in regulating the retrieval of CI-M6PR from endosomes to the trans-Golgi network (TGN) . Depletion of RUFY1 leads to a delay in CI-M6PR retrieval, resulting in impaired delivery of newly synthesized hydrolases to lysosomes . RUFY1 functions by mediating dynein-dependent retrograde transport of CI-M6PR-containing endosomes to the TGN . This process is essential for the efficient sorting of lysosomal hydrolases, as it enables the recycling of their receptor (CI-M6PR) back to the TGN where it can bind newly synthesized hydrolases for subsequent delivery to lysosomes .

How does the Arl8b-RUFY1 complex contribute to endosomal positioning and movement?

The Arl8b-RUFY1 complex functions at recycling endosomes, regulating the positioning and movement of these organelles . RUFY1 interacts with the dynein-dynactin complex through its coiled-coil region, facilitating dynein-dependent retrograde transport of endosomes toward the perinuclear region . This mechanism is similar to that of other dynein activating adaptors and is critical for proper endosomal trafficking and cargo sorting . The Arl8b-RUFY1 complex specifically functions on a subset of endosomes that morphologically resemble early endosomes and multivesicular bodies, as demonstrated by immuno-electron microscopy .

What other cargo sorting processes involve RUFY1?

Beyond CI-M6PR trafficking, RUFY1 has been implicated in several other cargo sorting processes. It regulates transferrin receptor and integrin recycling from early endosomes . Additionally, RUFY1 association with early endosomes increases following ligand-mediated activation of the epidermal growth factor receptor (EGFR), and RUFY1 knockdown results in prolonged retention of EGFR in early endosomal compartments . RUFY1, along with Rabenosyn-5, Rab4, and AP-3, also regulates early endosomal sorting of melanosomal cargo towards maturing melanosomes, which are lysosome-related organelles .

What are the optimal approaches for studying RUFY1-Arl8b interactions?

To study RUFY1-Arl8b interactions, researchers should consider the following approaches:

• GST pulldown assays: Use recombinantly purified GST-tagged Arl8b loaded with either GTP or GDP as bait to pull down FLAG-tagged RUFY1 isoforms from transfected cell lysates .

• Co-immunoprecipitation: Examine the interaction of endogenous RUFY1 and Arl8b by co-immunoprecipitation from cell lysates using specific antibodies .

• Direct protein-protein interaction assays: Utilize purified proteins (e.g., GST-RUFY1 RUN domain and His-tagged Arl8b) to assess direct binding in vitro .

• Site-directed mutagenesis: Generate and test Arl8b mutants (e.g., Q75L for constitutively GTP-bound form, T34N for constitutively GDP-bound form) and RUFY1 mutants (e.g., R206A/R208A for disrupted Arl8b binding) to dissect the specificity and requirements of the interaction .

What imaging techniques are most effective for visualizing RUFY1 localization and trafficking?

For optimal visualization of RUFY1 localization and trafficking, consider the following imaging techniques:

• Confocal microscopy: Use for colocalization studies with various endosomal markers (e.g., Rab14, EEA1, SNX1, CI-M6PR) .

• Structured illumination microscopy (SIM): Apply this super-resolution technique to resolve objects separated by 100-150 nm, allowing for more precise determination of RUFY1 localization relative to other proteins .

• Immuno-electron microscopy: Utilize for ultrastructural analysis of RUFY1-positive compartments and their morphological characteristics .

• Live-cell imaging: Implement to track the dynamics of RUFY1-positive endosomes and their interactions with other cellular compartments.

What are the critical controls for RUFY1 knockdown experiments?

When designing RUFY1 knockdown experiments, the following controls are essential:

• Validation of knockdown efficiency: Use Western blotting to confirm the depletion of both RUFY1 isoforms (~80 kD and ~70 kD) .

• Antibody specificity control: Ensure the specificity of RUFY1 antibodies by demonstrating the disappearance of target bands in siRNA-treated samples .

• Rescue experiments: Perform phenotype rescue by expressing siRNA-resistant RUFY1 constructs to confirm that observed effects are specifically due to RUFY1 depletion .

• Domain-specific mutants: Include experiments with RUFY1 mutants lacking specific domains (e.g., ΔRUN) or containing point mutations (e.g., R206A/R208A) to dissect domain-specific functions .

How can researchers investigate the role of RUFY1 in disease models?

To investigate RUFY1's role in disease models, researchers should consider:

• CRISPR/Cas9-mediated knockout: Generate RUFY1 knockout cell lines or animal models to assess phenotypic consequences in specific tissues or disease contexts.

• Disease-relevant cell types: Examine RUFY1 expression and function in cell types that are particularly relevant to diseases involving lysosomal dysfunction (e.g., neurons for neurodegenerative disorders).

• Patient-derived samples: Analyze RUFY1 expression, localization, and function in samples from patients with lysosomal storage disorders or other conditions potentially linked to endosomal trafficking defects.

• Correlation with disease markers: Investigate the relationship between RUFY1 dysfunction and established markers of disease progression or severity.

What are the current gaps in understanding RUFY1 function and potential research directions?

Current knowledge gaps and future research directions include:

• Tissue-specific functions: Investigating whether RUFY1 has distinct roles in different tissues or cell types beyond the commonly studied HeLa and HEK293T cells .

• Isoform-specific functions: Determining whether the longer and shorter isoforms of RUFY1 have distinct or overlapping functions .

• Regulation of RUFY1 activity: Exploring how RUFY1 function is regulated by post-translational modifications or other regulatory mechanisms.

• Interaction with other trafficking machinery: Investigating how RUFY1 coordinates with other components of the endosomal sorting machinery, such as the retromer complex .

• Role in other trafficking pathways: Examining RUFY1's potential involvement in additional trafficking pathways beyond CI-M6PR retrieval and lysosomal hydrolase delivery.