The Endoplasmic Reticulum (ER) Translocon Can Differentiate between Hydrophobic Sequences Allowing Signals for Glycosylphosphatidylinositol Anchor Addition to Be Fully Translocated into the ER Lumen*

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (ω) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.

[1]  V. Gurevich,et al.  Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. , 1991, Analytical biochemistry.

[2]  S. Udenfriend,et al.  [39] Processing of nascent proteins to glycosylphosphatidylinositol-anchored forms in cell-free systems , 1995 .

[3]  G. Hart,et al.  Rapid processing of the carboxyl terminus of a trypanosome variant surface glycoprotein. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Wang,et al.  Recognition of the carboxyl-terminal signal for GPI modification requires translocation of its hydrophobic domain across the ER membrane. , 1999, Journal of molecular biology.

[5]  S. High,et al.  Early Events in Glycosylphosphatidylinositol Anchor Addition , 2001, The Journal of Biological Chemistry.

[6]  S. High,et al.  The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells. , 1995, The Biochemical journal.

[7]  N. Singh,et al.  How to make a glycoinositol phospholipid anchor. , 1992, Trends in biochemical sciences.

[8]  M. Ferguson,et al.  The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. , 1999, Journal of cell science.

[9]  I. Imhof,et al.  Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins. , 2000, Biochemistry.

[10]  G von Heijne,et al.  Glycosylation Efficiency of Asn-Xaa-Thr Sequons Depends Both on the Distance from the C Terminus and on the Presence of a Downstream Transmembrane Segment* , 2000, The Journal of Biological Chemistry.

[11]  S. High,et al.  Mechanisms that determine the transmembrane disposition of proteins. , 1992 .

[12]  S. Udenfriend,et al.  An Active Carbonyl Formed during Glycosylphosphatidylinositol Addition to a Protein Is Evidence of Catalysis by a Transamidase (*) , 1995, The Journal of Biological Chemistry.

[13]  M. Aebi,et al.  Yeast Gpi8p is essential for GPI anchor attachment onto proteins. , 1996, The EMBO journal.

[14]  R. Greenspan,et al.  Conversion of placental alkaline phosphatase from a phosphatidylinositol-glycan-anchored protein to an integral transmembrane protein. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[15]  A. Menon,et al.  Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system. , 2001, European journal of biochemistry.

[16]  J. Bonifacino,et al.  Uncleaved signals for glycosylphosphatidylinositol anchoring cause retention of precursor proteins in the endoplasmic reticulum. , 1993, The Journal of biological chemistry.

[17]  N. Inoue,et al.  PIG‐S and PIG‐T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8 , 2001, The EMBO journal.

[18]  Jialing Lin,et al.  Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins. , 2003, Molecular cell.

[19]  Jialing Lin,et al.  Both Lumenal and Cytosolic Gating of the Aqueous ER Translocon Pore Are Regulated from Inside the Ribosome during Membrane Protein Integration , 1997, Cell.

[20]  Min Goo Lee,et al.  A protein sequence that can encode native structure by disfavoring alternate conformations , 2002, Nature Structural Biology.

[21]  H. Riezman,et al.  Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins. , 2000, Molecular biology of the cell.

[22]  S Udenfriend,et al.  How glycosylphosphatidylinositol-anchored membrane proteins are made. , 1995, Annual review of biochemistry.

[23]  K. Kodukula,et al.  Structural requirements of a nascent protein for processing to a PI-G anchored form: studies in intact cells and cell-free systems. , 1991, Cell biology international reports.

[24]  B. Wilkinson,et al.  Determination of the Transmembrane Topology of Yeast Sec61p, an Essential Component of the Endoplasmic Reticulum Translocation Complex* , 1996, The Journal of Biological Chemistry.

[25]  K. Kodukula,et al.  Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide. , 1991, The Journal of biological chemistry.

[26]  H. Riezman,et al.  Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins , 1995, The Journal of cell biology.

[27]  M. Ferguson,et al.  The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. , 1993, The Biochemical journal.

[28]  A. van Dorsselaer,et al.  The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p. , 2001, Molecular biology of the cell.

[29]  G. von Heijne,et al.  A Nascent Secretory Protein 5 Traverse the Ribosome/Endoplasmic Reticulum Translocase Complex as an Extended Chain (*) , 1996, The Journal of Biological Chemistry.

[30]  B. Martoglio,et al.  The protein-conducting channel in the membrane of the endoplasmic reticulum is open laterally toward the lipid bilayer , 1995, Cell.