Recombinant Danio rerio Protein FAM91A1 (fam91a1), partial

What is the molecular function of FAM91A1 in Danio rerio?

FAM91A1 functions as a component of the WDR11 complex and works together with TBC1D23 to facilitate the golgin-mediated capture of vesicles generated using AP-1 . It plays a critical role in endosome-to-Golgi trafficking pathways . FAM91A1 and TBC1D23 cooperate to regulate trafficking of KIAA0319L, a protein known to regulate axonal growth . The protein consists of 831 amino acids and adopts a unique structural fold with 18 alpha-helices and 2 beta-strands forming a triangular pyramid-like arrangement .

What phenotypes emerge when FAM91A1 is depleted in zebrafish models?

When FAM91A1 is depleted in zebrafish using splice-blocking morpholino technology, developmental defects highly similar to those observed with TBC1D23 depletion occur . Specifically:

• Significant reduction in FAM91A1 mRNA levels (>60% reduction) following injection of 5.0 ng of morpholino

• Reduced midbrain size by approximately 60% compared to control groups

• Abnormal CaP motor neuron morphology

• Reduced CaP axon length (only 67% of the length observed in control zebrafish)

These findings strongly suggest that FAM91A1 plays an essential role in neurodevelopment, particularly in brain and motor neuron development .

How does FAM91A1 interact with TBC1D23 and what is the functional significance?

FAM91A1 directly interacts with TBC1D23 through its N-terminal region (residues 1-328) . The interaction is characterized by:

• TBC1D23 binding to a conserved surface on FAM91A1 by assuming a Z-shaped conformation

• Multiple hydrogen bonds, salt bridges, and Van der Waals forces creating a buried surface area of ~1,003 Ų

• Three distinct interaction segments within TBC1D23 (residues 518-524, 525-531, and 532-538)

This interaction is essential for their cooperative function in protein trafficking from endosomes to the trans-Golgi network (TGN) . Disruption of this interaction correlates with developmental defects in zebrafish and potentially with human neurological disorders .

What techniques are optimal for studying FAM91A1 expression and interactions?

For protein detection and interaction studies:

For zebrafish studies:

• Splice-blocking morpholino technology (5.0 ng dose) achieves ~60% reduction in FAM91A1 mRNA levels

• Transgenic lines such as Tg[Hb9:GFP]ml2 for visualizing motor neurons

• Fluorescence microscopy for assessing midbrain development

What expression systems are effective for producing recombinant Danio rerio FAM91A1?

For structural studies:

• A chimera construct encoding TBC1D23 (residues 514-543) followed by a 3×GGS linker and FAM91A1 N-terminus (residues 1-328) has successfully produced diffraction-quality crystals

• Further truncation experiments show that TBC1D23 residues 514-538, but not 539-558, directly interact with FAM91A1 N-terminus

For functional studies:

• The N-terminal region of FAM91A1 (residues 1-328) is sufficient for interaction with TBC1D23

• When designing truncations or mutations, consider key residues involved in TBC1D23 binding (R61, R91, K190, D194, D198)

• Mutations in these residues can serve as negative controls for interaction studies:

  • K190A/D194A (KD AA) - completely abolishes binding

  • D198R - completely abolishes binding

  • R61A - decreases binding by >80%

How does FAM91A1 relate to human neurological disorders?

FAM91A1 has emerged as a potential pontocerebellar hypoplasia (PCH)-associated gene based on several lines of evidence:

• Mutations in WDR11, a subunit of the FAM91A1 complex, have been found in patients with PCH-like symptoms

• Depletion of FAM91A1 in zebrafish produces developmental defects similar to those seen with TBC1D23 depletion, and TBC1D23 is an established PCH-associated gene

• The FAM91A1-TBC1D23 interaction regulates endosome-to-Golgi trafficking of proteins critical for neuronal development, such as KIAA0319L

These findings suggest that impaired endosomal trafficking represents a convergent mechanism for many PCH subtypes .

What is known about the evolutionary conservation of FAM91A1?

Sequence analysis across model organisms reveals:

• Key residues involved in the FAM91A1-TBC1D23 interaction are highly conserved across diverse species including human, mouse, zebrafish, frog, fruit fly, and nematode

• This conservation suggests the functional importance of this interaction has been maintained throughout evolution

• The N-terminal domain of FAM91A1 adopts a novel fold that appears to be structurally conserved, supporting its critical functional role

This evolutionary conservation provides valuable insights for researchers designing experiments with recombinant zebrafish FAM91A1 and considering translational implications for human disease models.

What structural features characterize the FAM91A1-TBC1D23 complex?

The crystal structure of the FAM91A1-TBC1D23 complex reveals:

• FAM91A1 N-terminus (residues 1-328) adopts a novel fold not identified by DALI structural similarity searches

• The structure encompasses 18 alpha-helices and 2 beta-strands, forming a triangular pyramid-like arrangement:

  • α1–6 form the first cone

  • α7–11 form the second cone

  • α12–15 and β1–2 form the third cone

  • α16–18 form the fourth cone

• TBC1D23 (residues 518-538) assumes a Z-shape and binds to the surface between the first, second, and third cones

• Residues 236-251 of FAM91A1 likely form a structurally flexible loop not visible in the electron density

How do specific mutations affect FAM91A1-TBC1D23 binding?

Detailed molecular analysis of the interaction interface reveals:

• First segment (TBC1D23 residues 518-524): H519 forms hydrogen bonds with N217 and T93 in FAM91A1; D524 forms a salt bridge and hydrogen bond with FAM91A1 R91

• Second segment (TBC1D23 residues 525-531): Y530 forms two hydrogen bonds with FAM91A1 K190 and D194; R531 contacts FAM91A1 D198 via salt bridge and hydrogen bond

• Mutations in critical FAM91A1 residues (K190A/D194A or D198R) completely abolish binding to TBC1D23, while R61A decreases binding by over 80%

Understanding these specific interactions provides valuable targets for structure-based design of experiments to probe FAM91A1 function or potentially develop therapeutic approaches for related disorders.

What controls are essential when studying recombinant FAM91A1 function?

When investigating FAM91A1 function, researchers should include:

• Positive controls: A549 cells express detectable levels of FAM91A1 and serve as suitable positive controls for Western blot experiments

• Negative controls: FAM91A1 mutants (K190A/D194A, D198R, or R61A) that disrupt TBC1D23 binding can serve as functional negative controls

• Rescue experiments: Wild-type FAM91A1 expression should rescue phenotypes observed in FAM91A1-depleted models if effects are specific

• Interaction partners: Consider examining effects on known interacting proteins including C3H17orf75, TBC1D23, BET1, CPDA, GOSR1, DSCC1, AKAP1b, GOSR2, ATAD2, and SCFD1

How can researchers quantitatively assess FAM91A1-dependent trafficking?

To measure FAM91A1's role in endosome-to-Golgi trafficking:

• Monitor fluorescently-tagged cargo proteins such as KIAA0319L

• Assess colocalization with trans-Golgi network markers

• Measure morphological changes in motor neurons using Tg[Hb9:GFP]ml2 transgenic zebrafish lines

• Quantify developmental phenotypes like midbrain size reduction (~60% in FAM91A1-depleted zebrafish) and CaP axon length reduction (~33%)

These quantitative measurements provide robust readouts for FAM91A1 function in trafficking and development.