Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis

Abstract

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-␣ (TGF␣), Naked2 coats TGF␣-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGF␣-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na ؉ /K ؉ -ATPase ␣1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles.

Myristoylated Naked2 Antagonizes Wnt-β-Catenin Activity by Degrading Dishevelled-1 at the Plasma Membrane

“…NKD2 clearly interacts with both Dvl-1 and TGF␣, but we have never seen a ternary complex, leading to the model we propose below. Moreover, none of the Dvls were found among the 389 proteins identified in NKD2-associated exocytic vesicles by liquid chromatography-tandem mass spectrometry (13).…”

“…Cell Fractionation-The plasma membrane, vesicles, and other subcellular organelles in Caco-2-TGF␣ cells were separated using an Iodixanol gradient fractionation method as described previously (13). Briefly, Caco-2-TGF␣ cells were washed and lysed in Solution E (0.25 M sucrose, 78 mM KCl, 4 mM MgCl 2 , 8 mM CaCl 2 , 10 mM EGTA, and 50 mM Hepes-KOH, pH 7.0).…”

“…3D). To assess biochemically where endogenous NKD2/DVL-1 interact, cell fractionation studies were performed in TGF␣-expressing Caco-2 cells using a discontinuous 10 -40% iodixanol gradient, a strategy we recently employed to enrich biochemically and then flow sort a plasma membrane-depleted pool of G2A NKD2 vesicles for liquid chromatography-tandem mass spectrometry analysis (13). The early fractions in the gradient were enriched for plasma membrane constituents as demonstrated by the migration patterns of E-cadherin and Na ϩ /K ϩ ATPase ␣1.…”

Hijacking of RIG-I Signaling Proteins into Virus-Induced Cytoplasmic Structures Correlates with the Inhibition of Type I Interferon Responses

“…For visualization of SFTSV NSs cytoplasmic vesicles, we modified the protocol of Cao et al (47). Briefly, HeLa cells expressing SFTSV NSs fused to mCherry were rinsed three times in PBS, followed by one rinse in solution D (78 mM KCl, 4 mM MgCl 2 , 8 mM CaCl 2 , 10 mM EDTA, 50 mM HEPES-KOH, pH 7.0).…”

A Monoclonal Antibody Produced Against Naked2

“…(1,2) NKD2, with a molecular mass of 50 kDa, is predominantly localized on the vesicles or plasma membrane. (3) Full-length NKD2 and its 1-331 fragment are poorly soluble.…”