Acid prohormone sequence determines size, shape, and docking of secretory vesicles in atrial myocytes.
How vesicles are born in the trans-Golgi network and reach their docking sites at the plasma membrane is still largely unknown and is investigated in the present study on live, primary cultured atrial cardiomyocytes. Secretory vesicles (n=422) are visualized by expressing fusion proteins of proatrial natriuretic peptide (proANP) and green fluorescent protein. Myocytes expressing fusion proteins with intact proANP display two populations of fluorescent vesicles with apparent diameters of 120 and 175 nm, moving at a top velocity of 0.3 microm/s. The number of docked vesicles is significantly correlated with the number of mobile vesicles (r=0.71, P<0.0005). The deletion of the acidic N-terminal proANP[1-44] or point mutations (glu(23,24)-->gln(23,24)) change size and shape-but not velocity-of the vesicles, and, strikingly, abolish their docking at the plasma membrane. The shapes thus change from spheres to larger, irregular floppy bags or vesicle trains. Deletion of the C-terminal proANP[45-127], where the ANP and its disulfide bond reside, does not change size, shape, docking, or velocity of the mobile vesicles. The N-terminal acid calcium-binding sequence of proANP is known to cause protein aggregation at the high calcium concentration prevailing in the trans-Golgi network. Therefore, these results indicate that amino acid residues favoring cargo aggregation are critically important in shaping the secretory vesicles and determining their fate-docking or not docking-at the plasma membrane. The full text of this article is available at http://www.circresaha.org.
Baertschi
A J
AJ
Department of Physiology, Centre Médical Universitaire, University of Geneva, Geneva, Switzerland. alex.baertschi@medecine.unige.ch
Monnier
D
D
Schmidt
U
U
Levitan
E S
ES
Fakan
S
S
Roatti
A
A
D000818:Animals; D000831:Animals, Newborn; D009320:Atrial Natriuretic Factor / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D049452:Green Fluorescent Proteins; D006325:Heart Atria / Q000378:metabolism* / Q000648:ultrastructure; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016253:Microscopy, Immunoelectron; D008863:Microspheres; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism* / Q000648:ultrastructure; D010316:Particle Size; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D022142:Secretory Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction / Q000502:physiology; D013329:Structure-Activity Relationship; D021601:trans-Golgi Network / Q000378:metabolism
D008164:Luminescent Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D009320:Atrial Natriuretic Factor; D002118:Calcium