The Major Yolk Protein of Sea Urchins Is Endocytosed by a Dynamin-Dependent Mechanism1

Abstract

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.

Dysferlin is essential for endocytosis in the sea star oocyte

“…We could envision a feedback system whereby if exocytosis is reduced by the dysferlin knock-down, then endocytosis would be reduced to retain total plasma membrane surface area. Endocytosis is a critical process utilized by growing oocytes to import yolk protein precursors (Brooks and Wessel, 2004; Opresko and Wiley, 1987). In C.elegans , endocytosis of cell surface proteins was also found to be critical for the regulation of meiotic maturation in oocytes (Cheng et al, 2008).…”

Complexity of Yolk Proteins and Their Dynamics in the Sea Star Patiria miniata

“…It is also important to know when and where MYP originated within the phylum. By virtue of the presence of MYP seen here in the early germ line (the posterior enterocoel) and the fact that it is not utilized early in development in the sea urchins tested (Brooks and Wessel, 2002, Brooks and Wessel, 2003, Brooks and Wessel, 2004), suggests that MYP may be doing something different than vitellogenin, even though it is stored in a similar granule. The diversity of developmental and reproductive strategies within echinoderms makes this phylum an excellent resource for these broad, important questions.…”

“…MYP protein in sea urchins is made in the adult gut, transported to the ovary (likely by diffusion) and up taken by somatic cells that transport the protein to the oocyte, which endocytoses it via a dynamin-dependent mechanism (Brooks and Wessel, 2002, Brooks and Wessel, 2003, Brooks and Wessel, 2004). The lack of mRNA expression in sea star oocytes does not exclude the possibility that MYP protein is actually present in the oocytes, since MYP may also be synthesized primarily in extra-gonadal tissue and taken up into sea star oocytes much like seen in the sea urchin.…”

“…Typically, large yolk protein precursors are synthesized in extragonadal tissues (Opresko and Wiley, 1987, Raikhel and Dhadialla, 1992, Reimer and Crawford, 1995, Voogt et al ., 1985, Wallace and Selman, 1981) and in the somatic cells that support the developing gametes (Majewska et al ., 2014, Reunov et al ., 2010, Unuma et al ., 2007, Unuma et al ., 2010, Unuma et al ., 2003). These precursors are then proteolytically cleaved into smaller yolk proteins while within cells in the gonad (Brooks and Wessel, 2002, Brooks and Wessel, 2003, Brooks and Wessel, 2004, Prowse and Byrne, 2012, Yamamura et al ., 1995). For most oviparous invertebrate and vertebrate animals, the primary large yolk protein precursor is Vitellogenin (Vtg; (Yamamura et al ., 1995)), a glycophospholipoprotein belonging to the Large Lipid Transfer Protein (LLTP) superfamily (Babin et al ., 1999, Smolenaars et al ., 2007) .…”

“…Because Vtg is the primary yolk protein in invertebrates, it may have been inferred in earlier studies on echinoderms that the major protein of the yolk granules (major yolk protein (MYP)) is a Vtg. However, the sea urchin MYP is not a Vtg, but rather is a transferrin-like protein that can bind metals such as iron and zinc (Brooks and Wessel, 2002, Brooks and Wessel, 2003, Brooks and Wessel, 2004, Unuma et al ., 2007). Further, the MYP of sea urchins persists throughout development, a result inconsistent with its presumed role in nutrition for gametogenesis or development.…”