Journal of Molecular Biology
Volume 286, Issue 5, 12 March 1999, Pages 1303-1310
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Communication
Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane1

https://doi.org/10.1006/jmbi.1999.2584Get rights and content

Abstract

A carboxyl-terminal hydrophobic domain is an essential component of the processed signal for attachment of the glycosyl-phosphatidylinositol (GPI) membrane anchor to proteins and it is linked to the site (ω) of GPI modification by a spacer domain. This study was designed to test the hypothesis that the hydrophobic domain interacts with the lipid bilayer of the endoplasmic reticulum (ER) membrane to optimally position the ω site for GPI modification. The hydrophobic domain of the GPI signal in the human folate receptor (FR) type α was substituted with the carboxyl-terminal segment of the low-density lipoprotein receptor (LDLR), including its membrane spanning region, without altering either the spacer or the ω site. The FR-α/LDLR chimera was not GPI modified but was attached to the plasma membrane by a polypeptide anchor. When the carboxyl-terminal half of the hydrophobic transmembrane polypeptide in the FR-α/LDLR chimera was altered by introduction of negatively charged (Asp) residues, or when the cytosolic domain in the chimera was deleted, the mutated proteins became GPI-anchored. On the other hand, attachment of a carboxyl-terminal segment of LDLR including the entire cytosolic domain to FR-α converted it into a transmembrane protein. The results indicate that in the FR-α/LDLR chimera the inability of the cellular machinery for GPI modification to recognize the hydrophobic domain is not due to the intrinsic nature of the peptide, but is rather due to the retention of the peptide within the lipid bilayer. It follows that the hydrophobic domain in the signal for GPI modification must traverse the ER membrane prior to recognition of the ω site by the GPI-protein transamidase. The results thus establish a critical topographical requirement for recognition of the GPI signal in the ER.

Section snippets

Replacement of the hydrophobic domain of the GPI signal in FR-α with the carboxyl-terminal sequence of the LDL receptor

A moderately hydrophobic domain is a common feature of GPI signals. There exists a hydrophobicity threshold for this domain below which the function of the GPI signal is impaired Lowe 1992, Nuoffer et al 1993, Yan and Ratnam 1995. However, there is no evidence that an increase in the hydrophobicity of this peptide would change the efficiency of GPI modification. We constructed cDNA encoding a chimeric protein in which the carboxyl-terminal hydrophobic domain of FR-α was replaced by the

Conversion of the transmembrane polypeptide of FR-α1-243/LDLR768-839to a GPI signal by introduction of charged residues or deletion of the cytoplasmic peptide

Integral membrane proteins employ the same translocation machinery for membrane insertion as secretory proteins use to cross the membrane (Von Heijne, 1997). There are two important sequence requirements to keep a protein stably integrated in the lipid bilayer: a hydrophobic segment that spans the membrane followed by positively charged residues Blobel 1980, Sabatini et al 1982. Most integral membrane proteins have at least one hydrophobic segment consisting of about 20 amino acid residues,

Conversion of FR-α to a transmembrane protein by attachment of a carboxyl-terminal segment of LDLR

These results would predict that if the carboxyl-terminal GPI signal of FR-α were somehow stabilized in the lipid bilayer of the ER, the protein would not be modified by GPI. To test this prediction, a cDNA construct was made in which, the carboxyl-terminal peptide, residues 783 to 839 of LDLR was attached to the carboxyl terminus of FR-α. The carboxyl-terminal segment includes the entire cytosolic domain of LDLR and seven upstream hydrophobic residues (Figure 1). The resulting fusion protein,

Location of the site of GPI signal recognition in the ER

The results of this study contradict the original hypothesis that the hydrophobic domain of the GPI signal interacts with components of the ER membrane to optimally position the ω site for the action of GPI-protein transamidase. That hypothesis was based in part on the close similarities of the hydrophobic domain of the GPI signal with that of the leader peptide (Yan & Ratnam, 1995). In the latter case it was proposed that the hydrophobic domain is anchored within the lipid bilayer to

Acknowledgements

The authors thank Mrs Jenny Zak for typing the manuscript.

This research is Supported by NCI grant R29CA5-7598.

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