Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a 
frequent cause of autosomal recessive parkinsonism and are also involved in 
sporadic Parkinson's disease. Loss of Parkin function is thought to compromise 
the polyubiquitylation and proteasomal degradation of specific substrates, 
leading to their deleterious accumulation. Several studies have analyzed the 
effects of parkin gene mutations on the biochemical properties of the protein. 
However, the absence of a cell-free system for studying intrinsic Parkin 
activity has limited the interpretation of these studies. Here we describe the 
biochemical characterization of Parkin and 10 pathogenic variants carrying 
amino-acid substitutions throughout the sequence. Mutations in the RING fingers 
or the ubiquitin-like domain decreased the solubility of the protein in 
detergent and increased its tendency to form visible aggregates. None of the 
mutations studied compromised the binding of Parkin to a series of known protein 
partners/substrates. Moreover, only two variants with substitutions of conserved 
cysteine residues of the second RING finger were inactive in a purely in vitro 
ubiquitylation assay, demonstrating that loss of ligase activity is a minor 
pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed 
the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein 
ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, 
in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate 
p38, rather than its polyubiquitylation. Thus, Parkin may mediate 
polyubiquitylation or proteasome-independent monoubiquitylation depending on the 
protein context. The discovery of monoubiquitylated Parkin species in cells 
hints at a novel post-translational modification potentially involved in the 
regulation of Parkin function.