A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase. Academic Article uri icon

Overview

MeSH

  • Amino Acid Sequence
  • Animals
  • Conserved Sequence
  • DNA, Complementary
  • Flow Cytometry
  • HeLa Cells
  • Hexosaminidases
  • Humans
  • Mannose
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Point Mutation
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Transfection

MeSH Major

  • Acyltransferases
  • Membrane Glycoproteins
  • Proline

abstract

  • Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.

publication date

  • February 20, 2004

has subject area

  • Acyltransferases
  • Amino Acid Sequence
  • Animals
  • Conserved Sequence
  • DNA, Complementary
  • Flow Cytometry
  • HeLa Cells
  • Hexosaminidases
  • Humans
  • Mannose
  • Membrane Glycoproteins
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Point Mutation
  • Precipitin Tests
  • Proline
  • Protein Binding
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Transfection

Research

keywords

  • Journal Article

Identity

Language

  • eng

Digital Object Identifier (DOI)

  • 10.1074/jbc.M312191200

PubMed ID

  • 14660601

Additional Document Info

start page

  • 6540

end page

  • 6545

volume

  • 279

number

  • 8