ABSTRACT
Glycosylphosphatidylinositol (GPI)-anchored proteins are generally absent from the surface of cells that are defective in GPI biosynthesis. The current study was undertaken to: (a) examine in detail the intracellular localization and fate of precursors of GPI-anchored proteins in cells that fail to add GPI groups and (b) define structural characteristics of the precursor proteins that determine their intracellular localization. By examining GPI-deficient cells, we show that the uncleaved precursor of the GPI-anchored protein, Q7b, is retained in the cisternae of the endoplasmic reticulum (ER) and is largely lost intracellularly with a half-time of 2-4 h. Only a small amount (1-10%) of a proteolytically cleaved form of the protein is secreted into the medium. In cells competent for GPI anchor addition, mutation of the putative cleavage/attachment site for GPI addition in Q7b results in a similar phenotype of ER retention of the uncleaved precursor. An aspartic acid residue (Asp316) within the Q7b GPI anchoring signal, previously found to be essential for GPI anchor addition (Waneck, G. L., Stein, M. E., and Flavell, R. A. (1988) Science 241, 697-699), is also shown to be critical for ER retention. Information leading to ER retention is transferable to another protein leading to ER retention is transferable to another protein by fusion of the GPI anchoring signals from either Q7b or the GPI-anchored form of the IgG Fc receptor type III. Analysis by sedimentation on sucrose gradients shows that Q7b species retained in the ER are multimeric, whereas species that exit the ER are monomeric. This correlation suggests that the presence of an uncleaved signal for GPI anchoring induces changes in the aggregation state of the precursor proteins, which may lead to their retention in the ER.
