A selective pathway for degradation of cytosolic proteins by lysosomes.

A lysosomal pathway of proteolysis is selective for cellular proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent cultured cells that are deprived of serum growth factors and in certain tissues of fasted animals. We have reconstituted this lysosomal degradation pathway in vitro. Transport into lysosomes requires a KFERQ-like sequence in the substrate protein and uptake and/or degradation is stimulated by ATP. A member of the heat shock 70 kDa protein family, the 73 kDa constitutive heat shock protein, binds to KFERQ-like peptide regions within proteins and, in some as yet unidentified manner, facilitates transfer of the proteins into lysosomes. Several possible mechanisms of selective protein transport into lysosomes are discussed.

Efficient saturation mutagenesis of a pentapeptide coding sequence using mixed oligonucleotides.

Site-directed mutagenesis using oligonucleotides that are degenerate at a specific codon was employed to construct a set of mutations in a pentapeptide sequence targeting cytosolic proteins to lysosomes during serum withdrawal. Low-temperature annealing of the mixed oligonucleotides to single-stranded phage DNA and a genetic selection for the DNA strand carrying the mutations were utilized. The use of mixed oligonucleotides by this technique provides an economical means of generating a large set of substitution mutations. A single codon can be changed to codons for most other amino acids in one step. This approach eliminates the need for restriction enzyme cleavage sites flanking the target for mutagenesis and, therefore, is useful for targeting mutations to any DNA fragment cloned into an appropriate single-stranded bacteriophage.