Recombinant Mouse Protrudin (Zfyve27)

What is Protrudin (Zfyve27) and what is its cellular localization?

Protrudin (Zfyve27) is a novel member of the FYVE-finger family of proteins that was originally identified as an interacting partner of spastin, which is frequently mutated in hereditary spastic paraplegia . The protein contains several functional domains including a Rab11 binding domain (RBD11) in its N-terminal region, a FYVE domain in its C-terminal end, a FFAT motif, a coiled-coil domain, and three hydrophobic region (HR) motifs in the central portion of the protein . These structural features are characteristic of proteins involved in membrane-cargo trafficking.

Subcellular localization studies using epitope-tagged constructs (E2-ZFYVE27 or GFP-ZFYVE27) demonstrate that Protrudin is predominantly expressed in punctate vesicles within cells . Biochemical analyses including subcellular fractionation and Triton X-114 membrane phase separation indicate that ZFYVE27 is a peripheral membrane protein that binds to phosphatidylinositol 3-phosphate lipid moiety .

How does Protrudin contribute to neurite formation?

Protrudin plays a crucial role in promoting neurite extension through directional membrane trafficking . It acts in concert with Rab11, a small GTPase that regulates membrane traffic at the trans-Golgi network-recycling endosome boundary and recycles them back to the plasma membrane . This coordinated activity is essential for directional membrane transport during neurite formation.

Experimental evidence shows that overexpression of ZFYVE27 in PC12 cell lines and primary hippocampal neurons leads to extensive neurite outgrowth . Conversely, downregulation of endogenous ZFYVE27 in PC12 cells by RNA interference results in inhibition of neurite outgrowth even after nerve growth factor induction and causes swelling of cell soma . These findings demonstrate that Protrudin is a critical determinant of neuronal differentiation and development.

What is the significance of Protrudin oligomerization?

Protrudin forms functional oligomers, specifically dimers or tetramers, which are essential for its biological activity . The oligomerization of ZFYVE27 was initially discovered through a yeast two-hybrid screen that identified ZFYVE27 as its own interaction partner . This self-interaction was subsequently confirmed in mammalian cells using co-immunoprecipitation and co-localization studies .

Sucrose gradient centrifugation experiments revealed that ZFYVE27 oligomerizes into dimer/tetramer forms . The core interaction region between Protrudin monomers was mapped to the third hydrophobic region (HR3, amino acids 185-207) of the protein through deletion construct analysis in yeast two-hybrid assays . Importantly, cells expressing ZFYVE27 with deleted HR3 motif (ZFYVE27 ΔHR3) fail to produce cellular protrusions and instead exhibit swelling of cell soma, indicating that oligomerization is necessary for Protrudin's ability to promote neurite extensions .

What disease associations have been found for Protrudin mutations?

Mutations in ZFYVE27 have been linked to hereditary spastic paraplegia (HSP), specifically the autosomal dominant form (AD-HSP) . A German family with AD-HSP was found to have a mutation in ZFYVE27, designated as SPG33 . The mutated ZFYVE27 protein shows an aberrant intracellular pattern in its tubular structure, and its interaction with spastin is severely affected .

The specific mutation in ZFYVE27 is postulated to affect neuronal intracellular trafficking in the corticospinal tract, which is consistent with the pathology of HSP . This finding connects Protrudin dysfunction with impaired axonal transport, which has been implicated in numerous neurodegenerative disorders. The SPG33 form appears to be a pure form of HSP, and ZFYVE27 has been excluded as a candidate gene for the SPG27 subtype of the disease .

What experimental approaches can be used to study Protrudin oligomerization?

Several complementary techniques can be employed to study Protrudin oligomerization:

Yeast Two-Hybrid Analysis:

• Generate a bait construct with full-length human ZFYVE27 cDNA cloned into appropriate vectors (e.g., pGBKT7)

• Transform into yeast strain (e.g., AH109) and test for auto-activation of reporter genes

• Perform direct-Y2H assay with ZFYVE27 constructs to confirm self-interaction

• Create deletion constructs to map interaction domains

Co-immunoprecipitation in Mammalian Cells:

• Create epitope-tagged ZFYVE27 constructs (e.g., GFP-ZFYVE27, FLAG-ZFYVE27)

• Co-transfect differentially tagged constructs into mammalian cells

• Perform immunoprecipitation with tag-specific antibodies

• Analyze co-precipitated proteins by Western blotting

Sucrose Gradient Centrifugation:

• Prepare cell lysates containing ZFYVE27

• Subject lysates to sucrose gradient centrifugation (e.g., 5-20% sucrose gradient)

• Collect fractions and analyze by Western blotting

• Compare migration patterns with known molecular weight markers to determine oligomeric state

Fluorescence Resonance Energy Transfer (FRET):

• Generate fluorescent protein-tagged ZFYVE27 constructs (e.g., CFP-ZFYVE27, YFP-ZFYVE27)

• Perform live-cell imaging to detect energy transfer between fluorophores

• Calculate FRET efficiency to quantify protein-protein interactions

How can researchers effectively analyze Protrudin membrane association?

To investigate Protrudin's association with membranes, researchers can utilize these methodological approaches:

Subcellular Fractionation:

• Homogenize cells expressing ZFYVE27 in appropriate buffer

• Separate cellular components by differential centrifugation

• Analyze distribution of ZFYVE27 across cytosolic, membrane, and nuclear fractions by Western blotting

Triton X-114 Membrane Phase Separation:

• Treat cell lysates with Triton X-114 at 4°C

• Warm samples to 37°C to induce phase separation

• Separate aqueous and detergent phases

• Analyze presence of ZFYVE27 in each phase to determine membrane association properties

Lipid Binding Assays:

• Prepare liposomes containing various phosphoinositides

• Incubate with purified recombinant ZFYVE27

• Pellet liposomes and analyze bound proteins

• Particularly focus on phosphatidylinositol 3-phosphate binding via the FYVE domain

Confocal Microscopy with Membrane Markers:

• Co-express fluorescently tagged ZFYVE27 with established membrane markers

• Perform live-cell or fixed-cell confocal microscopy

• Quantify colocalization using appropriate image analysis software

What methods are optimal for analyzing the effects of Protrudin mutants?

When investigating Protrudin mutants, particularly those lacking the HR3 domain or containing disease-associated mutations, consider these approaches:

Dominant-Negative Effect Analysis:

• Co-express wild-type ZFYVE27 with mutant variants (e.g., ZFYVE27 ΔHR3)

• Assess cellular phenotypes using microscopy techniques

• Quantify changes in neurite extension, cell morphology, and protein localization

• Analyze cytoplasmic swelling as a readout of disrupted function

Neurite Outgrowth Assays:

• Express ZFYVE27 variants in neuronal cell lines (e.g., PC12) or primary neurons

• Induce differentiation (e.g., with nerve growth factor for PC12 cells)

• Quantify neurite length, number, and branching pattern

• Compare mutant phenotypes to wild-type and negative controls

Interaction Studies with Spastin:

• Perform co-immunoprecipitation of ZFYVE27 variants with spastin

• Quantify interaction efficiency using Western blotting

• Utilize FRET or proximity ligation assays to assess interactions in intact cells

• Compare binding of disease-associated mutants to determine functional consequences

Live-Cell Trafficking Analysis:

• Generate fluorescently tagged ZFYVE27 variants

• Perform time-lapse microscopy to track vesicle movement

• Analyze trafficking dynamics including velocity, directionality, and processivity

• Correlate trafficking defects with cellular phenotypes

How can researchers design experiments to study Protrudin in neuronal systems?

For effective neuronal studies of Protrudin function, consider these methodological approaches:

Primary Neuronal Cultures:

• Isolate and culture primary neurons (hippocampal, cortical, or motor neurons)

• Transfect or transduce neurons with ZFYVE27 constructs

• Analyze neurite outgrowth, branching, and neuronal morphology

• Implement time-lapse imaging to track dynamic changes in neurite extension

In Vivo Models:

• Generate transgenic mouse models with ZFYVE27 modifications (knockout, knockin of specific mutations)

• Analyze corticospinal tract development and maintenance

• Assess motor function using behavioral assays relevant to HSP

• Perform histological analyses of neuronal morphology and axonal integrity

Rab11 Interaction Studies:

• Co-express ZFYVE27 with wild-type and mutant Rab11 (constitutively active or dominant negative)

• Analyze effects on ZFYVE27 localization and function

• Quantify changes in vesicle trafficking and neurite extension

• Identify key regulatory mechanisms in the ZFYVE27-Rab11 pathway

Axonal Transport Assays:

• Express fluorescently tagged cargo proteins in neurons with modified ZFYVE27 expression

• Perform live-cell imaging of cargo transport along axons

• Quantify transport parameters (velocity, run length, pause frequency)

• Correlate transport defects with neuronal development and maintenance

What techniques can be used to purify recombinant Protrudin for structural and functional studies?

For researchers working with recombinant Protrudin, these purification and analysis methods are recommended:

Expression Systems:

• Use prokaryotic systems (E. coli) for expression of soluble domains

• Employ eukaryotic systems (insect cells, mammalian cells) for full-length protein

• Consider cell-free systems for proteins that may be toxic to host cells

• Optimize expression conditions to maximize protein yield and solubility

Purification Strategy:

• Design constructs with appropriate tags (His, GST, MBP) to facilitate purification

• Implement multi-step purification protocol including affinity chromatography, ion exchange, and size exclusion

• Verify protein identity by mass spectrometry

• Assess protein quality using dynamic light scattering and thermal shift assays

Functional Validation:

• Perform lipid binding assays to confirm interaction with phosphatidylinositol 3-phosphate

• Analyze oligomerization state using analytical ultracentrifugation or multi-angle light scattering

• Conduct in vitro interaction studies with binding partners including spastin and Rab11

• Validate functional activity through reconstitution assays in membrane systems

Structural Analysis:

• Apply X-ray crystallography or cryo-electron microscopy for high-resolution structure determination

• Use small-angle X-ray scattering (SAXS) to analyze oligomeric assemblies in solution

• Implement nuclear magnetic resonance (NMR) for dynamic studies of specific domains

• Apply hydrogen-deuterium exchange mass spectrometry to map protein-protein interaction interfaces