In this report we demonstrated the existence of an Ufm1 conjugation pathway in the human protozoan parasite Leishmania donovani including the enzymatic steps involving activation by Uba5 (E1), conjugation by Ufc1 (E2) and finally conjugation with substrate proteins. Modification and/or alterations in the expression of Ufm1 and Uba5 showed parasite stage specific growth effects underlining the importance of Ufm1 mediated protein modifications in Leishmania growth and differentiation. Importantly all the components of Ufm1 conjugation and the substrate proteins in Leishmania appear to be associated with the mitochondria. To our knowledge this is the first demonstration of a trypanosomatid mitochondrial associated complete Ubl pathway in which the activation, conjugation components and the substrate proteins are all localized in mitochondria.
Initial studies showed that the Ufm1 pathway is widely conserved in mammals, nematode and other multicellular organisms but absent in unicellular eukaryotes such as yeast 
. Our first of a kind demonstration of Ufm1 pathways in trypanosomatid parasites, unicellular eukaryotes that emerged as early eukaryotic organisms indicates the ancient origins of this pathway. In trypanosomatid parasites, our analysis showed that the Ubls such as Ub, SUMO and Ufm1 appear to have one or more aminoacid residues after the C-terminal glycine that need to be processed before conjugation (data not shown). Among the trypanosomatids, only Leishmania
Ufm1 has a longer C-terminal extension that is 17 residues long. However, our results suggest that in L. donovani
C-terminal processing of Ufm1 does occur suggesting the presence of a processing protease and the Gly98 residue is essential for that cleavage and subsequent conjugation. In contrast, apicomplexan parasites such as Plasmodium
contain Ubls (Atg8) that terminate in a C-terminal Glycine indicating the absence of processing prior to conjugation 
. This structural diversity among different parasites might be a result of adaptations that parasites have undergone in their respective environments during their complex life cycles.
Similar to humans, LdUba5 interacts with LdUfm1 and activates it in an ATP dependent reaction. Also, biochemical characteristics shared between human Uba5 and LdUba5 and activation of human Ufm1 by LdUba5 demonstrated the mechanistic similarities between the pathways in the two species. Previously it has been demonstrated that human Ufm1 interacts with its activating enzyme Uba5 involving the cysteine residue in the active site 
. Also, it has been shown in many of the E1 and E1-like enzymes of Ub or Ubls that changing the cysteine residue in the active site to serine results in formation of stable intermediates between the E1 and its respective modifier protein. Our studies showed that LdUba5C217S
mutant also makes a stable intermediate with LdUfm1. The formation of stable intermediate is likely due to the covalent ester link between Uba5 and Ufm1 as previously observed in other E1-like enzymes 
. It is reasonable to expect overexpression of LdUba5C217S
would have a negative dominant effect which will result in depletion of the free pool of endogenous Ufm1. Interestingly, overexpression of the LdUba5C217S
mutant resulted in significant reduction of L. donovani
amastigote growth suggesting a role of LdUfm1 conjugation in parasite pathogenesis (). Similarly, protein interactions between LdUfm1 and LdUfc1 showed the conservation of E2-like activity in L. donovani
parasites, thereby demonstrating the importance of E2 activity in the Leishmania
Ufm1 conjugation pathway as well as E1 activity. Recently, human E3-ligase Ufl1 that conjugates Ufm1 to its substrate protein was identified 
. Database searches in the trypanosomatid genomes indicated that these parasites may encode an ortholog of human ufl1, albeit with low sequence homology. However, E3 ligase independent conjugation of Ubls via E2 activity is still a distinct possibility as was recently reported 
One of the primary reasons for the inability to assign any biological function to the Ufm1 pathway in any organism so far is that the target proteins that are conjugated by Ufm1 have not been characterized. Our LC/MS results showed that LdUfm1 is potentially conjugated to the 40S ribosomal protein SA and the mitochondrial trifunctional protein α-subunit. It is known that the 40S ribosomal protein SA is required for the assembly and/or stability of the 40S ribosomal subunit. In L. tarentolae
40S ribosomal subunits have been shown to be enriched in mitochondria 
. A role for Ubls in the regulation of either stability or activity of mammalian ribosomal proteins was indicated by their conjugation to NEDD 
The mitochondrial trifunctional protein catalyzes three consecutive reactions in the β-oxidation of long-chain fatty acids, and plays important role in control and regulation of β-oxidation 
. Fatty acid oxidation is an essential energy generation system that occurs in mitochondria. In T. brucei
, a trypanosomatid parasite related to Leishmania
, proteomic analysis revealed that the mitochondrial trifunctional protein is enriched in the mitochondria 
. In trypanosomatids the regulation of β-oxidation of long chain fatty acids is not completely understood. Further studies should reveal if conjugation with Ufm1 has any role in altering the activity or signaling the translocation of the mitochondrial trifunctional protein during this energy generation process. Recently, a protein with a suggested role in membrane transport was reported as the first identified target of human Ufm1 modification 
Our results showed that in L. donovani
Ufm1, Uba5, and Ufc1 are found associated with the mitochondrion. In addition, both the targets for Ufm1 conjugation that we identified in this study also are associated with the mitochondrion. The presence of the components of Leishmania
Ufm1 pathway and its substrates argue for the existence of a mitochondria associated Ufm1 conjugation pathway. In contrast, subcellular localization of human Ufm1as well as other components i.e., Uba5, Ufc1 or Ufl1 is not known 
. Previously E1, E2 of Ub or E3 of SUMO were shown independently to be associated with mitochondria in metazoans 
. To our knowledge, this is the first report that demonstrates the presence of complete Ufm1 pathway along with its substrates in the mitochondria.
Mitochondrion associated Ufm1 conjugation in Leishmania
presents several interesting possibilities regarding biological functions of this pathway. For example, ubiquitin involvement in the degradation of mitochondrial matrix proteins is known 
. Stability of several mammalian mitochondrial proteins is affected by proteasomal inhibition suggesting a role for ubiquitination 
. Several E3 ligases located on the outer mitochondrial membrane have been identified recently that primarily affect the mitochondrial morphology by either ubiquitination 
or by Sumoylation of mammalian proteins that regulate mitochondrial fission 
. It would be of interest to investigate whether the changes mediated by the Ufm1 conjugation have any impact on the life-cycle related differentiation of the parasites.
Demonstration of the existence of mitochondria associated Ufm1 conjugation in unicellular trypanosomatid parasites offers unique possibilities to explore the importance of this pathway in the context of its pathogenesis and host-parasite adaptations. Importantly, the ubiquitin-dependent proteolysis system (UPS) is increasingly recognized as a viable therapeutic pathway in the treatment of cancer after the successful treatment of hematological malignancies with proteasome inhibitors 
. Deubiquitinases, the key effectors of UPS and intracellular signaling cascades, and Ub ligases because of their narrow substrate specificity are emerging as important targets for potential anticancer therapies 
. Identification of Ufm1 mediated protein modification pathways in Leishmania
, with its distinct subset of substrate proteins associated with mitochondrial activities as demonstrated in this report may provide specific targets for novel drug therapies against this human pathogen.