Use of fluorescence-activated vesicle sorting for isolation of Naked2-associated, basolaterally targeted exocytic vesicles for proteomics analysis.
By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor-alpha (TGFalpha), Naked2 coats TGFalpha-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 TGFalpha-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 alpha1 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. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.
Cao
Zheng
Z
Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
Li
Cunxi
C
Higginbotham
James N
JN
Franklin
Jeffrey L
JL
Tabb
David L
DL
Graves-Deal
Ramona
R
Hill
Salisha
S
Cheek
Kristin
K
Jerome
W Gray
WG
Lapierre
Lynne A
LA
Goldenring
James R
JR
Ham
Amy-Joan L
AJ
Coffey
Robert J
RJ
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D015153:Blotting, Western; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D004285:Dogs; D005089:Exocytosis; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D021381:Protein Transport / Q000528:radiation effects; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism* / Q000528:radiation effects; D014283:Triiodobenzoic Acids / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C449092:NKD2 protein, human; D011506:Proteins; D016211:Transforming Growth Factor alpha; D014283:Triiodobenzoic Acids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C044834:iodixanol