Retention and degradation of proteins containing an uncleaved glycosylphosphatidylinositol signal.
Glycosylphosphatidylinositol (GPI) membrane anchor attachment is directed by a COOH-terminal signal that is proteolytically removed and replaced with a preformed GPI anchor in a coupled reaction. Failure to complete proteolytic cleavage and anchor addition results in the retention of an uncleaved precursor in a post-endoplasmic reticulum (ER) compartment. In this report, we address three issues: (i) the exact position of the transport block, (ii) the subsequent fate of the retained molecules, i.e. where are they degraded, and (iii) the mechanism whereby these proteins are selected for retention. Using decay accelerating factor (DAF), we provide evidence that failure to cleave the GPI signal totally prevents O-glycosylation, suggesting that the uncleaved polypeptides are not transported into the cis-Golgi complex. This implies that transport is blocked at the boundary between the ER-Golgi intermediate compartment and the Golgi stacks. The degradation of an intracellularly retained human growth hormone (hGH)-DAF fusion protein containing a nonfunctional GPI signal shows some features of ER degradation, i.e. the degradation is insensitive to leupeptin, chloroquine, and ammonium chloride, and is inhibited at 16 degrees C or after ATP depletion. However, morphological evidence points to a pathway resembling autophagy. To reconcile these observations, we suggest either that hGHDAF is degraded by two distinct pathways (ER degradation and autophagy) or that ER degradation takes place in an ER-associated vesicular compartment in a process resembling autophagy. Using as probes a soluble hGH receptor and an antibody recognizing only native hGH, we show that a significant fraction of the retained protein is correctly folded, ruling out general misfolding as the basis for retention. We also show that hGHDAF fusion proteins are present in high molecular weight, disulfide-linked aggregates in COS cells. We suggest a model for retention in which the uncleaved GPI signal drives the formation of large micelle-like aggregates that cannot be secreted.
Field
M C
MC
Department of Neurobiology, Genentech Inc., South San Francisco, California 94080.
Moran
P
P
Li
W
W
Keller
G A
GA
Caras
I W
IW
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D018958:CD55 Antigens; D016466:CHO Cells; D002402:Cathepsin D / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015703:Antigens, CD; D018958:CD55 Antigens; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D013006:Growth Hormone; D002402:Cathepsin D