The major yolk protein of sea urchins is endocytosed by a dynamin-dependent mechanism.
Sea urchin oocytes grow to 10 times their original size during oogenesis by both synthesizing and importing a specific repertoire of proteins to drive fertilization and early embryogenesis. During the vitellogenic growth period, the major yolk protein (MYP), a transferrin-like protein, is synthesized in the gut, transported into the ovary, and actively endocytosed by the oocytes. Here, we begin to dissect this mechanism by first testing the hypothesis that MYP endocytosis is dynamin-dependent. We have identified a sea urchin dynamin cDNA that is highly similar in amino acid sequence, structure, and size to mammalian dynamin I: it contains an N-terminal GTPase domain, a pleckstrin-homology domain, and a C-terminal proline-rich domain. Sea urchin dynamin is enriched at the cortex of oocytes and colocalizes to MYP endocytic vesicles at the oocyte periphery. To test for a functional relationship between MYP endocytosis and dynamin, we used a dominant-negative human dynamin I mutant protein containing an alteration within the GTPase domain (hDyn(K44A)) to specifically compete for dynamin function. Using a fluorescent MYP construct to follow its endocytosis solely, as well as a general endocytosis marker, we demonstrate that the disruption of dynamin function significantly reduces MYP uptake but does not affect fluid-phase endocytosis. Using this specific biochemical approach, we are able to separate distinct pathways of endocytosis during oogenesis and learn that dynamin-mediated endocytosis is responsible for MYP endocytosis but not fluid-phase uptake.
Brooks
Jacqueline M
JM
Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island 02912, USA.
Wessel
Gary M
GM
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D034281:Dynamins / Q000235:genetics / Q000378:metabolism*; D004527:Egg Proteins / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D020558:GTP Phosphohydrolases / Q000235:genetics; D047331:Lytechinus / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism
D004527:Egg Proteins; D020558:GTP Phosphohydrolases; D034281:Dynamins