Differential sorting and Golgi export requirements for raft-associated and raft-independent apical proteins along the biosynthetic pathway.
Sorting signals for apically destined proteins are highly diverse and can be present within the luminal, membrane-associated, and cytoplasmic domains of these proteins. A subset of apical proteins partition into detergent-resistant membranes, and the association of these proteins with glycolipid-enriched microdomains or lipid rafts may be important for their proper targeting. Recently, we observed that raft-associated and raft-independent apical proteins take different routes to the apical surface of polarized Madin-Darby canine kidney cells (Cresawn, K. O., Potter, B. A., Oztan, A., Guerriero, C. J., Ihrke, G., Goldenring, J. R., Apodaca, G., and Weisz, O. A. (2007) EMBO J. 26, 3737-3748). Here we reconstituted in vitro the export of raft-associated and raft-independent markers staged intracellularly at 19 degrees C. Surprisingly, whereas release of the raft-associated protein influenza hemagglutinin was dependent on the addition of an ATP-regenerating system and cytosol, release of a yellow fluorescent protein (YFP)-tagged raft-independent protein (the 75-kDa neurotrophin receptor; YFP-p75) was efficient even in the absence of these constituents. Subsequent studies suggested that YFP-p75 is released from the trans-Golgi network in fragile tubules that do not withstand isolation procedures. Moreover, immunofluorescence analysis revealed that hemagglutinin and YFP-p75 segregate into distinct subdomains of the Golgi complex at 19 degrees C. Our data suggest that raft-associated and raft-independent proteins accumulate at distinct intracellular sites upon low temperature staging, and that upon warming, they exit these compartments in transport carriers that have very different membrane characteristics and morphologies.
Guerriero
Christopher J
CJ
Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Lai
Yumei
Y
Weisz
Ora A
OA
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D015398:Signal Transduction; D013395:Sucrose / Q000737:chemistry; D013696:Temperature
D013395:Sucrose; D000255:Adenosine Triphosphate