Recombinant Mouse Phosphatidylinositide phosphatase SAC2 (Inpp5f), partial

What is the primary function of SAC2/INPP5F?

SAC2/INPP5F is a phosphoinositide 4-phosphatase that specifically hydrolyzes phosphatidylinositol 4-phosphate (PI4P). It functions primarily in the endocytic pathway, where it plays a crucial role in the sequential dephosphorylation of phosphoinositides. The enzyme contributes to phosphoinositide regulation by converting PI4P to phosphatidylinositol, which is an essential step in maintaining the proper phosphoinositide balance on endocytic membranes. This enzymatic activity suggests that SAC2/INPP5F helps orchestrate the conversion of membrane-specific phosphoinositides during vesicle trafficking, particularly in the progression from PI(4,5)P2-enriched plasma membrane to PI3P-positive endosomes .

Where is SAC2/INPP5F predominantly localized in cells?

SAC2/INPP5F exhibits a punctate distribution in the cell with significant localization to specific compartments of the endocytic pathway. Studies using GFP-tagged SAC2 in COS7 cells revealed that approximately 76.7% of GFP-Sac2 colocalized with endogenous Rab5, a marker for early endosomes . Furthermore, 54.2% of GFP-Sac2 colocalized with RFP-APPL1, which marks very early endosomal stations upstream of PI3P-positive endosomes . Importantly, SAC2/INPP5F shows minimal colocalization (only 3.51%) with the lysosomal marker LAMP1, indicating that its primary function is restricted to early stations of the endocytic pathway rather than later degradative compartments .

How does SAC2/INPP5F contribute to the endocytic recycling pathway?

SAC2/INPP5F plays a regulatory role in endocytic recycling, particularly for transferrin receptor (TfnR) trafficking. Experimental evidence demonstrates that disruption of SAC2 function affects both the steady-state distribution of TfnR and the kinetics of transferrin recycling. In cells expressing catalytically inactive SAC2 (SAC2CS), surface levels of TfnR are reduced by approximately 30% compared to control cells . Pulse-chase flow cytometry assays show that within the first 10 minutes, transferrin fluorescent signal in control cells decreases to 45.8%, while in cells expressing GFP-SAC2CS, the signal only decreases to 65.3%, indicating delayed recycling . Similar recycling defects are observed in SAC2 knockout cells generated using CRISPR technology, and these defects can be rescued by reexpression of wild-type SAC2 .

How does SAC2/INPP5F interact with other proteins in phosphoinositide regulation?

SAC2/INPP5F operates in coordination with other phosphoinositide-modifying enzymes to regulate membrane lipid composition during endocytosis. Research has demonstrated that SAC2/INPP5F colocalizes with OCRL (Oculocerebrorenal syndrome of Lowe) on endocytic membranes, including clathrin-coated vesicles, macropinosomes, and Rab5-positive endosomes . This relationship is particularly significant because OCRL is a 5-phosphatase that hydrolyzes PI(4,5)P2 to PI4P, which then becomes the substrate for SAC2/INPP5F. The interaction between OCRL and SAC2/INPP5F has been demonstrated by coimmunoprecipitation and is enhanced by Rab5, which is required for recruiting SAC2/INPP5F to endosomes . This sequential action of first OCRL and then SAC2/INPP5F mirrors the structure of synaptojanin, which contains both 5-phosphatase and 4-phosphatase (Sac domain) modules in a single protein .

What is the role of SAC2/INPP5F in STAT3 signaling pathways?

SAC2/INPP5F functions as an inhibitor of Signal Transducer and Activator of Transcription 3 (STAT3) signaling. Gene Set Enrichment Analysis (GSEA) has revealed that the STAT3 pathway is strongly correlated with INPP5F expression . Glioblastoma cells overexpressing INPP5F show reduced STAT3 activation, while cells with INPP5F knockdown exhibit increased STAT3 activation . Mechanistically, coimmunoprecipitation experiments demonstrate that INPP5F directly interacts with STAT3 in glioblastoma cell lysates . Further investigation using mutant STAT3 constructs has identified that INPP5F interacts specifically with the coiled-coil domain of STAT3 . This interaction affects STAT3 phosphorylation, which is a crucial step for STAT3 dimerization, nuclear translocation, and transcriptional activation of target genes .

How does the structure of SAC2/INPP5F relate to its function?

SAC2/INPP5F contains a catalytic Sac domain that defines its phosphatase activity. Structural characterization has revealed a unique pleckstrin-like homology Sac2 domain that is conserved across all Sac2 orthologues . The specificity of SAC2/INPP5F for PI4P is consistent with its strong similarity to the Sac domain of the Sac1 protein and to the catalytically active Sac domains of synaptojanins . For nuclear-cytoplasmic shuttling, SAC2/INPP5F contains nuclear localization signals (NLSs) and nuclear export signals (NESs) in its amino acid sequence, which can be predicted using bioinformatic tools . These structural elements enable SAC2/INPP5F to localize appropriately within the cell to execute its phosphatase function at the right time and place.

What are the key experimental methods for studying SAC2/INPP5F localization?

Studying SAC2/INPP5F localization requires a combination of molecular biology and imaging techniques. Fluorescent protein tagging, particularly GFP-tagging of SAC2/INPP5F, has been successfully employed to visualize its cellular distribution . When expressing tagged constructs, it is important to select cells with low/moderate expression levels to avoid artifacts from overexpression . Colocalization studies with established markers such as Rab5 (early endosomes), APPL1 (very early endosomes), EEA1 (PI3P-positive endosomes), and LAMP1 (lysosomes) can be performed using immunofluorescence or co-expression of fluorescently tagged markers .

For quantitative assessment of colocalization, researchers have reported that 76.7% of GFP-Sac2 colocalizes with endogenous Rab5, 80.0% of mCherry-Sac2 colocalizes with GFP-Rab5, and 54.2% of GFP-Sac2 colocalizes with RFP-APPL1 . These measurements provide important benchmarks for comparative studies.

How can knockout and knockdown models of SAC2/INPP5F be generated?

Multiple approaches have been employed to generate SAC2/INPP5F-deficient models for functional studies:

• CRISPR-Cas9 genome editing: This technique has been successfully used to generate SAC2 null cell lines with complete disruption of expression . The efficiency of knockout can be verified through Western blotting to confirm the absence of the protein.

• Mouse knockout models: Mouse embryonic fibroblasts (MEFs) have been derived from previously described Sac2/INPP5F knockout mice . These provide a valuable tool for studying the effects of SAC2/INPP5F deficiency in a physiologically relevant context.

• Expression of dominant-negative mutants: The catalytically inactive mutant Sac2C458S (Sac2CS) acts as a dominant-negative construct and can be used to disrupt SAC2/INPP5F function in transfected cells .

• RNA interference: Knockdown approaches using siRNA or shRNA targeting INPP5F can provide partial reduction of expression for studying dose-dependent effects.

What assays can measure SAC2/INPP5F enzymatic activity?

To assess the phosphatase activity of SAC2/INPP5F, several biochemical approaches can be employed:

• In vitro phosphatase assays: These directly measure the ability of purified SAC2/INPP5F to hydrolyze PI4P to phosphatidylinositol. The assay confirms that SAC2/INPP5F specifically functions as a 4-phosphatase rather than a 5-phosphatase .

• HPLC analysis of phosphoinositide levels: High-performance liquid chromatography of metabolically labeled cells can detect changes in steady-state levels of phosphoinositides. In SAC2 knockout MEFs, this approach revealed a minor (~5%) increase in PI4P levels compared to wild-type controls, suggesting functional redundancy with other PI 4-phosphatases .

• Fluorescent phosphoinositide reporters: Fluorescently tagged protein domains with specific phosphoinositide binding can be used to visualize changes in phosphoinositide distribution in living cells.

What is the connection between SAC2/INPP5F and Parkinson's disease?

SAC2/INPP5F has been identified as a risk locus in Parkinson's disease based on genetic studies . While the search results do not provide detailed mechanisms linking SAC2/INPP5F to Parkinson's pathophysiology, its function in endocytic pathways may be relevant since dysregulation of endosomal trafficking is increasingly recognized as a contributor to neurodegenerative diseases. The identification of SAC2/INPP5F as a risk factor suggests that alterations in phosphoinositide metabolism may contribute to Parkinson's disease pathogenesis, potentially through effects on protein degradation, autophagy, or synaptic vesicle recycling in neurons .

How does SAC2/INPP5F function in glioblastoma?

SAC2/INPP5F has been implicated in glioblastoma (GBM) through its regulation of STAT3 signaling. Studies show that INPP5F is differentially expressed in glioma stem-like cells (GSCs) from glioma patients . Functionally, INPP5F inhibits STAT3 signaling by directly interacting with STAT3 and inhibiting its phosphorylation . Constitutive expression of INPP5F suppresses self-renewal and proliferation potentials of glioblastoma cells and reduces tumorigenicity .

Gene expression microarray analysis identified 59 genes significantly regulated by INPP5F in both overexpressing and knockdown cells compared to controls . These findings suggest that SAC2/INPP5F may act as a tumor suppressor in glioblastoma by inhibiting the pro-oncogenic STAT3 pathway, making it a potential therapeutic target for this aggressive brain tumor.

What cellular phenotypes result from SAC2/INPP5F deficiency?

The absence of SAC2/INPP5F leads to several observable cellular phenotypes:

• Endocytic recycling defects: SAC2/INPP5F knockout cells show delayed recycling of transferrin and integrin . In pulse-chase assays, SAC2 null cells retain higher levels of internalized transferrin compared to control cells, indicating impaired recycling kinetics .

• Altered receptor distribution: SAC2 knockout or dominant-negative expression results in reduced surface levels of transferrin receptor despite unchanged total receptor amounts, suggesting a shift in the steady-state distribution from the plasma membrane to intracellular compartments .

• Cell migration defects: Genomic ablation of SAC2 causes defects in cell migration, potentially related to the observed impairment in integrin recycling .

• Phosphoinositide imbalance: While the steady-state levels of phosphoinositides show only minor changes in SAC2 knockout cells, there is likely a transient accumulation of PI4P on early endosomes that affects endosomal function .

How can structure-function relationships in SAC2/INPP5F be investigated?

Investigating structure-function relationships in SAC2/INPP5F involves several complementary approaches:

• Mutation and truncation analysis: Specific domains can be mutated or deleted to assess their roles in localization and function. For example, predictive bioinformatic tools can identify nuclear localization signals (NLSs) and nuclear export signals (NESs) in the amino acid sequence, which can then be mutated to alter cellular localization . When constructing NESs mutations, replacing leucine with alanine in predicted NESs can eliminate nuclear export ability .

• Domain mapping for protein interactions: Interaction domains can be identified using a series of truncated constructs. For example, studies with STAT3 mutants revealed that INPP5F interacts with full-length and N-terminal domain deleted STAT3 (131-End) but not with other truncations, demonstrating that INPP5F directly interacts with the coiled-coil domain of STAT3 .

• Catalytic site mutations: The Sac2C458S mutation creates a catalytically inactive protein that can be used to distinguish between phosphatase-dependent and phosphatase-independent functions .

What methodological considerations are important when analyzing SAC2/INPP5F in Transmembrane Recycling?

When analyzing SAC2/INPP5F's role in transmembrane receptor recycling, several methodological considerations are critical:

• Surface biotinylation assays: Surface proteins can be biotinylated with Sulfo-NHS-SS-biotin at 4°C (to halt endocytosis), isolated using streptavidin agarose beads, and quantified by Western blot. This approach allows measurement of surface-to-total ratio of receptors like transferrin receptor (TfnR) .

• Pulse-chase flow cytometry: Cells can be pulsed with fluorescently labeled ligands (e.g., Alexa Fluor 647-Transferrin) and chased for various time periods to quantify recycling kinetics. This method should include both wild-type controls and rescue experiments with re-expression of functional SAC2/INPP5F to confirm specificity .

• Confocal microscopy visualization: Direct visualization of internalized fluorescent cargo at different chase timepoints provides spatial information about recycling defects. In SAC2 null cells, prominent retention of intracellular transferrin signals is observed at later time points, indicating delayed recycling .

• Temperature controls: Endocytosis should be halted at 4°C for surface measurements, while recycling assays are typically performed at 37°C.

How might SAC2/INPP5F be targeted for therapeutic development?

Based on its functions in disease-relevant pathways, SAC2/INPP5F represents a potential therapeutic target: