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Membrane cofactor protein (CD46) of complement. Processing differences related to alternatively spliced cytoplasmic
M K Liszewski1, I Tedja, J P Atkinson
1Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Abstract:
Membrane cofactor protein (MCP, CD46), a widely distributed regulatory protein, inhibits complement activation on host cells and serves as a measles virus receptor. Most cells express four isoforms (with one of two cytoplasmic tails, CYT-1 or CYT-2). Previously, we noted that MCP precursors had variable intracellular processing. Therefore, we characterized the intracellular transport of individual MCP isoforms. Transfectants were used for pulse-chase analyses. MCP isoforms bearing CYT-1 chased into their mature, surface forms with a half-life (t1/2) of 10-13 min while those with CYT-2 required 35-40 min. The precursor of a tail-less mutant possessed a t1/2 of 160-165 min. Chimeras were constructed that added both tails in opposite orientation onto the isoform (i.e. CYT 1 + 2 or CYT 2 + 1). Chimera 1 + 2 precursor processed with a t1/2 of 35-37 min, similar to CYT-2. Chimera 2 + 1 had a t1/2 of 15-19 min, more closely resembling CYT-1. Thus, in both cases the carboxyl-terminal tail controlled the processing rate. Deletions were made in the beginning, middle, and carboxyl terminus of CYT-1. Deletion of the first or middle six amino acids had no effect on the processing rate. However, deletion of the terminal tetrapeptide (FTSL) slowed the rate to 30-32 min, suggesting that this sequence facilitates exit from the endoplasmic reticulum.
Insights
The carboxyl-terminal tail of Membrane Cofactor Protein (MCP, CD46) isoforms dictates their intracellular transport rate. Specific sequences within the tail, particularly the FTSL tetrapeptide, are crucial for efficient endoplasmic reticulum exit.
Area of Science:
- Immunology
- Cell Biology
- Virology
Background:
- Membrane Cofactor Protein (MCP, CD46) is a regulatory protein involved in complement inhibition and measles virus entry.
- MCP exists in four isoforms, differing in their cytoplasmic tails (CYT-1 and CYT-2).
- Previous observations indicated variable intracellular processing of MCP precursors.
Purpose of the Study:
- To characterize the intracellular transport and processing of individual MCP isoforms.
- To determine the role of cytoplasmic tails in MCP intracellular trafficking.
- To identify specific sequences within the MCP tail that regulate endoplasmic reticulum exit.
Main Methods:
- Pulse-chase analyses were performed on cells expressing different MCP isoforms and mutants.
- Chimera constructs were created to assess the influence of tail orientation on processing.
- Site-directed mutagenesis, including deletions within the CYT-1 tail, was employed.
Main Results:
- MCP isoforms with CYT-1 tails exhibited faster maturation (t1/2: 10-13 min) compared to CYT-2 tails (t1/2: 35-40 min).
- Tail-less MCP precursors showed significantly reduced processing rates (t1/2: 160-165 min).
- Deletion of the terminal FTSL sequence in CYT-1 significantly slowed processing (t1/2: 30-32 min), indicating its role in ER exit.
Conclusions:
- The carboxyl-terminal tail is the primary determinant of MCP intracellular processing rate.
- The FTSL tetrapeptide at the C-terminus of CYT-1 facilitates efficient exit from the endoplasmic reticulum.
- Understanding MCP trafficking is crucial for its regulatory functions and as a viral receptor.