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The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus
D S Leaf1, S J Roberts, J C Gerhart
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Abstract:
Protein secretion is blocked in Xenopus oocytes arrested at second meiotic metaphase. In this report, we show that secretion becomes blocked coincident with germinal vesicle breakdown (GVBD). Transport through the metaphase-arrested oocyte's secretory pathway continues unimpeded until proteins reach the trans-Golgi. These conclusions are drawn from experiments using exogenous prolactin and vesicular stomatitis virus G protein (VSV G) encoded by SP6 transcripts and endogenous glycosaminoglycan (GAG) chains initiated on beta-D-4-methylumbelliferyl-xyloside. From the initiation of maturation with progesterone until GVBD, secretion of prolactin synthesized before the start of maturation is comparable to secretion in immature oocytes, but after GVBD secretion of prolactin declines approximately 63% in the first hour. Not all steps in the secretory pathway are blocked when oocytes mature. Since VSV G protein acquires resistance to endo H digestion with equal efficiency in immature oocytes (arrested in first meiotic prophase) and matured oocytes (arrested in second meiotic metaphase), we conclude that transport of this protein from the ER to the Golgi is not inhibited at meiotic metaphase. Using [35S]sulfate to label xyloside-initiated GAG chains we find that transport of GAG chains from the trans-Golgi to the cell surface is 15-fold lower in matured oocytes than in immature oocytes. Examination of the size of GAG chains by SDS-PAGE and HPLC indicates that matured oocytes produce GAG chains significantly larger than GAG chains from immature oocytes. This increase in size suggests that GAG chains from matured oocytes have a longer residence time in the trans-Golgi than GAG chains from immature oocytes. Hence, part of the block to secretion in metaphase-arrested oocytes could be an inhibition of vesicle budding from the trans-Golgi.
Insights
Protein secretion is blocked in Xenopus oocytes during meiotic arrest. This block coincides with germinal vesicle breakdown (GVBD), affecting transport past the trans-Golgi and potentially inhibiting vesicle budding.
Area of Science:
- Cell Biology
- Developmental Biology
- Molecular Biology
Background:
- Xenopus oocytes are a model system for studying cell cycle regulation and protein secretion.
- Meiotic maturation involves significant cellular rearrangements and regulatory changes.
- Understanding protein secretion blockages is crucial for cell development and function.
Purpose of the Study:
- To investigate the precise stage and mechanism of protein secretion blockage in Xenopus oocytes arrested at second meiotic metaphase.
- To determine which steps of the secretory pathway are affected during oocyte maturation.
- To elucidate the role of germinal vesicle breakdown (GVBD) in regulating protein secretion.
Main Methods:
- Tracking secretion of exogenous proteins like prolactin and vesicular stomatitis virus G protein (VSV G).
- Analyzing endogenous glycosaminoglycan (GAG) chain synthesis and transport using [35S]sulfate labeling.
- Assessing protein modifications (e.g., endo H digestion) and GAG chain size (SDS-PAGE, HPLC).
Main Results:
- Protein secretion blockage occurs coincident with germinal vesicle breakdown (GVBD).
- Transport from ER to Golgi is unaffected, as shown by VSV G protein processing.
- Transport of GAG chains from trans-Golgi to cell surface is significantly reduced (15-fold lower) in matured oocytes.
- GAG chains are larger in matured oocytes, suggesting prolonged residence time in the trans-Golgi.
Conclusions:
- The secretory block in metaphase-arrested Xenopus oocytes primarily affects later stages of the pathway, specifically post-trans-Golgi.
- Inhibition of vesicle budding from the trans-Golgi is a likely mechanism contributing to the secretion block.
- Oocyte maturation involves complex regulation of the secretory pathway impacting protein and GAG chain export.