Recycling of the 67-kDa elastin binding protein in arterial myocytes is imperative for secretion of tropoelastin.
We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab')2 fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A1, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A1 inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin.
Hinek
A
A
Division of Cardiovascular Research, Hospital for Sick Children, University of Toronto, Ontario, Canada.
Keeley
F W
FW
Callahan
J
J
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001011:Aorta / Q000166:cytology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D002645:Chickens; D002738:Chloroquine / Q000494:pharmacology; D004549:Elastin / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus; D015151:Immunoblotting; D018942:Macrolides; D008854:Microscopy, Electron; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D009924:Organ Culture Techniques; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012756:Sheep; D014333:Tropoelastin / Q000032:analysis / Q000096:biosynthesis* / Q000187:drug effects; D014633:Valine / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D018942:Macrolides; D011956:Receptors, Cell Surface; D014333:Tropoelastin; C054901:elastin-binding proteins; D002738:Chloroquine; C040929:bafilomycin A1; D004549:Elastin; D014633:Valine