Co-and post-translational events in the biogenesis of pig small intestinal aminopeptidase N
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Abstract:
The biogenesis of pig small intestinal aminopeptidase N (EC 3. 4. 11. 2) was studied by cell-free translation of intestinal mRNA and by labelling of organ cultured intestinal explants. In cell-free translation, the primary mRNA translation product of aminopeptidase N was a polypeptide of Mr 115,000. When translation was performed in the presence of dog pancreatic microsomes, a Mr 140,000 polypeptide was also observed. A polypeptide of Mr 115,000 was seen for the enzyme, purified from tunicamycin exposed explants. This result suggests that aminopeptidase N is co-translationally inserted into the membrane without cleavage of the signal. Pulse-chase labelling of explants gave the following results: 1. Immediately after a 10 min pulse with [35S] methionine, aminopeptidase N was detected in the Ca2+-precipitated membrane fraction. 2. The earliest detectable form of the enzyme, a polypeptide of Mr 140,000, was "high mannose" glycosylated as judged by its sensitivity to endoglycosidase H. After 40 min of chase, a re-glycosylation, yielding the mature form of Mr 166,000, occurred. 3. Aminopeptidase N was expressed at the microvillar membrane after 60-90 min of chase. Monensin inhibited the conversion from high mannose to complex glycosylation and the appearance of the enzyme in the microvillar membrane, indicating a role of the Golgi complex in these processes. Colchicine prevented aminopeptidase N from reaching the microvillar membrane, suggesting the involvement of microtubules in the transport.
Insights
The biogenesis of pig small intestinal aminopeptidase N involves co-translational membrane insertion and sequential glycosylation. This process, crucial for enzyme function, occurs via the Golgi complex and requires microtubule transport to the microvillar membrane.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Aminopeptidase N (APN) is a key enzyme in small intestinal digestion.
- Understanding the biogenesis of APN is essential for comprehending its physiological role.
Purpose of the Study:
- To elucidate the biogenesis pathway of pig small intestinal aminopeptidase N.
- To investigate the co-translational insertion, glycosylation, and transport of APN.
Main Methods:
- Cell-free translation of intestinal mRNA with and without dog pancreatic microsomes.
- Pulse-chase labeling of organ-cultured intestinal explants.
- Enzyme purification from tunicamycin-exposed explants and treatment with endoglycosidase H.
Main Results:
- APN undergoes co-translational insertion into the membrane without signal cleavage.
- The enzyme is initially "high mannose" glycosylated (Mr 140,000) and matures to Mr 166,000 through re-glycosylation.
- APN reaches the microvillar membrane after 60-90 minutes, a process dependent on the Golgi complex and microtubule transport.
Conclusions:
- Pig small intestinal aminopeptidase N biogenesis involves co-translational membrane insertion and complex post-translational modifications.
- The Golgi complex and microtubules play critical roles in the transport and maturation of APN to the microvillar membrane.