ATP-dependent protein degradation

In the initial experiments, we resolved reticulocyte lysates on DEAE-cellulose into two crude fractions Fraction 1, which contained proteins not adsorbed to the resin, and Fraction 2, which contained all proteins adsorbed to the resin and eluted with high salt. The original aim of this fractionation was to get rid of hemoglobin, which was known to be in Fraction 1, while most non-hemoglobin proteins of reticulocytes were known to be in Fraction 2. We found that neither fraction was active by itself, but ATP-dependent protein degradation could be reconstituted by combination of the two fractions [13]. The active component in Fraction 1 was a small, heat-stable protein we have exploited its stability to heat treatment for its purification to near homogeneity. We termed this protein at that time APF-1, for ATP-dependent Proteolysis Factor 1 [13]. The identity of APF-1 with ubiquitin was established later by Wilkinson et al. [14], subsequent to the discovery in my laboratory of its covalent ligation to protein substrates, as described below. 

Ubiquitin-dependent protein degradation pathway. ATP hydrolysis is required to attach ubiquitin to the carrier protein El, which then passes the ubiquitin moiety to the activating protein E2. The protein E3 recognizes the NH2-terminal residues and directs E2 to conjugate ubiquitin to lysine residues in the protein. 

Multiprotein complex that catalyses ATP-dependent degradation of proteins tagged with ubiquitin. 

Proteins are degraded by both ATP-dependent and ATP-independent pathways. Ubiquitin targets many intracellular proteins for degradation. Liver cell surface receptors bind and internalize circulating asialoglycoproteins destined for lysosomal degradation. 

Proteins which are destined for degradation by the proteosome are first modified by the enzyme-catalysed attachment of numerous molecules of the protein ubiquitin, through amino groups to the protein targeted for degradation. This marks out the protein for ATP-dependent hydrolysis by the 26S proteosome, releasing peptides and ubiquitin... 

The proteolytic activity of some multicatalytic peptidases is stimulated by ATP, whereas that of others is not influenced by ATP [32], The ATP-dependent proteolytic system first found in reticulocytes requires the presence of a heat-stable polypeptide called ubiquitin, one of the roles of which is to mark particular proteins for subsequent degradation [33. ATP-Indepen-dent multicatalytic peptidases can degrade proteins that have a free amino or an /V-acctylatcd terminus, as well as oxidatively altered or phosphorylat-ed proteins [34], The small peptides generated are resistant to multicatalytic peptidases and are further degraded by cytoplasmic endopeptidases and exopeptidases. 

Three other components that my laboratory has identified and partially purified from Fraction 2 of reticulocytes, termed CF1-CF3, are involved in the degradation of proteins ligated to ubiquitin [24]. These are apparently subcomplexes of the 26S proteasome, a large ATP-dependent protease complex first described by Re-chsteiner and co-workers [25], CF3 is identical to the 20S proteasome core particle [26], while CFl and CF2 may be similar to the base and lid subcomplexes of the 19S regulatory particle of the 26S proteasome, described more recently by the Finley laboratory [27], In hindsight, the reason for finding subcomplexes, rather than the complete 26S complex in Fraction 2 was technical we have routinely prepared Fraction 2 from ATP-depleted reticulocytes [20], under which conditions the 26S proteasome dissociates to its subcomplexes. We found that incubation of the three subcomplexes in the presence of ATP promotes their assembly to the 26S proteasome [24, 26]. The role of ATP in the assembly of the 26S proteasome complex remains unknown. 

Haas, A. L., and Heeshko, A. ATP-dependent conjugation of reticulocyte proteins with the polypeptide required for protein degradation. Proc. Natl. Acad. Sci. USA 1980, 77, 1365-68. 

Low expression levels and the lability of the HslVU complex make work with proteins from wild-type strains difficult. Gratifyingly, the active protease can be reconstituted in vitro from over-expressed and purified components (Rohrwild et al. 1996). It requires ATP for the degradation of folded substrates and ATP or some of its analogs for the purification of small chromogenic peptides. As expected, ATP-hydrolysis and proteolysis activities are mutually dependent (Seol et al. 1997). In addition, the peptidase activity was found to depend in complex ways on the presence of various cations, especially K in the buffers (Huang and Goldberg 1997). 

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