Protein mobilities and P-selectin storage in Weibel-Palade bodies.
Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.
Kiskin
Nikolai I
NI
Division of Molecular Neuroendocrinology, MRC National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. nkiskin@nimr.mrc.ac.uk
Hellen
Nicola
N
Babich
Victor
V
Hewlett
Lindsay
L
Knipe
Laura
L
Hannah
Matthew J
MJ
Carter
Tom
T
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D021381:Protein Transport; D060149:Tetraspanin 30; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D020302:Weibel-Palade Bodies / Q000187:drug effects / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000075745:rab27 GTP-Binding Proteins
D015703:Antigens, CD; C554586:CD63 protein, human; D008565:Membrane Proteins; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D060149:Tetraspanin 30; C485184:enhanced green fluorescent protein; D000075745:rab27 GTP-Binding Proteins; D000643:Ammonium Chloride; D049452:Green Fluorescent Proteins; D010959:Tissue Plasminogen Activator; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins