Ronald Hough

The selective degradation of injected proteins occurs principally in the cytosol rather than in lysosomes

These studies use microinjection to determine whether the selective degradation of cytosolic proteins involves selective transfer of proteins to lysosomes or selective proteolysis within the cytosol. 14C-Sucrose-labeled bovine serum albumin (14C-sucBSA) was conjugated to polylysine, and monolayers of L929 cells were exposed to the conjugate. The 14C-sucrose-labeled peptides that arose upon degradation of the added 14C-sucBSA polylysine accumulated exclusively within lysosomes. In contrast, when 14C-sucBSA or 14C-sucrose-labeled pyruvate kinase (14C-sucPK) was microinjected into L929 cells, over half the 14C-sucrose-labeled peptides derived form the injected proteins were present in the postlysosomal supernatant. Control experiments demonstrated that the microinjection procedure did not cause 14C-sucrose peptides to leak from lysosomes. Therefore, the presence in the cytosol of substantial amounts of the degradation products from injected 14C-sucBSA and 14C-sucPK confirms the existence of a major proteolytic system(s) within or readily accessible to the cytosol of animal cells.

Ubiquitin/ATP-Dependent Protease

In 1980, Hershko et al. 1 proposed that attachment of ubiquitin (Ub) to substrate proteins marks those proteins for destruction. The marking hypothesis, which is shown schematically in Figure 1, has received substantial support during the past seven years. Five studies have demonstrated a good correlation between ubiquitination of a protein and its rapid proteolysis. First, when hemoglobin is injected into cultured mammalian cells, which are then treated with phenylhydrazine, globin is rapidly degraded. The concentration of globin-ubiquitin conjugates that form upon denaturation of hemoglobin is proportional to the rate of globin degradation.2 Second, proteins that incorporate amino acid analogs are generally degraded rapidly, and the concentration of Ub conjugates increases in Ehrlich ascites cells exposed to analogs.3 Third, Dictyostelium calmodulin is ubiquitinated at lysine 115 and subsequently degraded after being added to reticulocyte lysate; bovine calmodulin, which contains a methylated lysine 115, is not conjugated to Ub and is more stable.4 Fourth, phytochrome, a cytoplasmic light receptor in plants, exists in two interconvertible forms that differ in half-life by more than 100-fold. When dark-grown oat seedlings receive a light-flash, rapid degradation of phytochrome follows. At the same time, a portion of the phytochrome molecules become multiply ubiquitinated.5 Fifth, it has been found that upon expression of ubiquitin-β-galactosidase fusion proteins in yeast, ubiquitin is rapidly removed from the N terminus of β-galactosidase.6 When site-directed mutagenesis is used to produce enzymes with different residues at the N terminus, the resulting proteins vary considerably in stability. Those with Met, Ser, Ala, Thr, Val, or Gly are stable; those with N-terminal Phe, Leu, Asp, Lys, or Arg were degraded with half-lives less than 3 min. Western blots show that the latter set of proteins are ubiquitinated, some with as many as 15 attached ubiquitins.6