Proteins that fail to fold in the endoplasmic reticulum (ER) or cannot find a 
pattern for assembly are often disposed of by a process named ER-associated 
degradation (ERAD), which involves transport of the substrate protein across the 
ER membrane (dislocation) followed by rapid proteasome-mediated proteolysis. 
Different ERAD substrates have been shown to be ubiquitinated during or soon 
after dislocation, and an active ubiquitination machinery has been found to be 
required for the dislocation of certain defective proteins. We have previously 
shown that, when expressed in tobacco (Nicotiana tabacum) protoplasts, the A 
chain of the heterodimeric toxin ricin is degraded by a pathway that closely 
resembles ERAD but is characterized by an unusual uncoupling between the 
dislocation and the degradation steps. Since lysine (Lys) residues are a major 
target for ubiquitination, we have investigated the effects of changing the Lys 
content on the retrotranslocation and degradation of ricin A chain in tobacco 
protoplasts. Here we show that modulating the number of Lys residues does not 
affect recognition events within the ER lumen nor the transport of the protein 
from this compartment to the cytosol. Rather, the introduced modifications have 
a clear impact on the degradation of the dislocated protein. While the 
substitution of the two Lys residues present in ricin A chain with arginine 
slowed down degradation, the introduction of four extra lysyl residues had an 
opposite effect and converted the ricin A chain to a standard ERAD substrate 
that is disposed via a process in which dislocation and degradation steps are 
tightly coupled.