Covalent linkage of ribonuclease S-peptide to microinjected proteins causes their intracellular degradation to be enhanced during serum withdrawal.

Abstract

The amino-terminal 20 amino acids are required for micro' jected ribonuclease A (RNase A) to be taken up by lysosomes and degraded at an enhanced rate during serum withdrawal. We used water-soluble carbodlimides to covalently attach the RNase S-peptide (residues 1-20) to [3H]RNase S-protein (residues 21-124) at unspecified locations. We then measured catabolism of the [3H]S-protein-S-peptide conjugate after its microiujection into human diploid fibroblasts. The attached S-peptide caused the degradation of S-protein to be enhanced 2-fold in the absence of serum.Control experiments showed that degradation of [3H]RNase S-protein remained unresponsive to serum after conjugation with the inactive fragment, RNase S-peptide (residues 1-10). Covalent attachment of RNase S-peptide had a similar effect on the catabolism of two other proteins. Degradation rates of microiijected '5I-labeled lysozyme and '251-labeled insulin A chain are normally unresponsive to serum withdrawal. However, breakdown rates of micro'mjected "SI-labeled lysozyme-S-peptide and '251-labeled insulin A chain-S-peptide conjugates were increased 2-fold during serum deprivation. We suggest that RNase S-peptide acts as a "signal sequence" that directs cytosolic proteins to lysosomes through a pathway that is activated by deprivation conditions. Multiple pathways of intracellular protein degradation exist in mammalian cells (1-4), and the current challenge is to define the roles of each pathway in contributing to overall proteolysis. For example, cytosolic ATP-dependent pathways of protein catabolism are responsible for the degradation of abnormal proteins (2, 3, 5, 6) and perhaps certain short-lived normal proteins (7,8), while lysosomes are responsible for most of the increased proteolysis in tissues of starved animals (9-12) and in cultured cells deprived of serum (1,13,14). Both lysosomal and nonlysosomal pathways probably contribute to the breakdown of intracellular proteins in optimally nourished tissues (2, 9-11) and in cultured cells supplemented with serum (1,14).Among the many advantages of erythrocyte-mediated microinjection (15-17) is the ability to modify proteins of interest prior to microinjection to probe effects of protein structure on rates and pathways of proteolysis. For example, several microinjection studies have addressed the role of ubiquitin in the rapid degradation of abnormal proteins by ATP-dependent pathways in the cytosol (6,(18)(19)(20).We have concentrated on pathways of degradation of long-lived proteins whose catabolic rates are increased in cultured cells in response to serum withdrawal (21-24). Pancreatic ribonuclease A (RNase A) microinjected into human fibroblasts is degraded with a half-life of 80-100 hr. When serum is removed from the fibroblasts, its degradative rate is increased 2-fold (22). The S-peptide region (residues 1-20 of RNase A) contains the information required for recognition of the entire protein for enhanced catabolism during serum withdrawal. RNase S-protein (residues of RNase A) is degrade...

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