Proteolytic cleavage of haptoglobin occurs in a subcompartment of the endoplasmic reticulum: evidence from membrane fusion in vitro.
The primary translation product of haptoglobin mRNA is a 45-kD polypeptide which is proteolytically cleaved shortly after its synthesis. Previous studies have indicated that the cleavage of this proform of haptoglobin occurs in the ER. In an attempt to characterize the cleaving enzyme, we found that upon incubation of microsomes from rat hepatocytes pulse labeled with [35S]methionine, little cleavage of labeled prohaptoglobin occurred. In contrast, when cells whose cytoplasmic proteins had been released by saponin treatment were incubated, 30-40% of the prohaptoglobin was cleaved. The addition of GTP caused a twofold stimulation, which was abolished by the nonhydrolyzable analog GTP gamma S. With a homogenate of the cells, the addition of GTP resulted in a fourfold stimulation of the degree of cleavage--from 15 to 60%. Differential centrifugation revealed that most of the cleaving activity resided in membranes sedimenting similarly to mitochondria and to a small fraction of the ER. These rapidly sedimenting membranes were therefore prepared from a rat liver homogenate. Upon treatment with high salt, light membranes were released which, when incubated with microsomes of pulse-labeled hepatocytes in the presence of detergent (and in the absence of GTP), induced specific cleavage of prohaptoglobin. The cleaving enzyme had an alkaline pH optimum indicating that it was not of lysosomal origin. These results suggest that cleavage of prohaptoglobin occurs in a subcompartment of the ER. Apparently, the connection between this compartment and the bulk of the ER is broken upon saponin treatment or homogenization but can be reestablished through a process requiring GTP hydrolysis.
Wassler
M
M
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Fries
E
E
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D003600:Cytosol / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006242:Haptoglobins / Q000032:analysis* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008099:Liver / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D008561:Membrane Fusion / Q000502:physiology*; D008715:Methionine / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism / Q000648:ultrastructure; D010447:Peptide Hydrolases / Q000502:physiology*; D011498:Protein Precursors / Q000378:metabolism; D051381:Rats; D012503:Saponins / Q000494:pharmacology; D013462:Sulfur Radioisotopes
D006242:Haptoglobins; D011498:Protein Precursors; D012503:Saponins; D013462:Sulfur Radioisotopes; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D008715:Methionine; D010447:Peptide Hydrolases