Characterization of functional domains in human Claspin

Claspin is a mediator of the ATR-dependent DNA replication checkpoint in human cells and also promotes DNA replication fork progression and stability. Though Claspin has been shown to bind DNA and co-immunoprecipitate with other replication fork-associated proteins, the specific protein-protein and protein-DNA interactions that are important for Claspin function are not known. We therefore purified several domains of human Claspin and then tested for direct interactions of these fragments with several replication fork-associated proteins and with DNA. Our data show that the N terminus of Claspin binds to the replicative helicase co-factor Cdc45, the Timeless protein and a branched, replication fork-like DNA structure. In contrast, the C terminus of Claspin associates with DNA polymerase epsilon and Rad17-Replication Factor C (RFC). We conclude that multiple protein-DNA and protein-protein interactions may be important for Claspin function during DNA replication and DNA replication checkpoint signaling.Key words: Claspin, DNA replication, checkpoint, DNA damage, Cdc45, DNA polymerase, Rad17     

Characterization of functional domains of human EB1 family proteins

EB1 family proteins are evolutionarily conserved proteins that bind microtubule plus-ends and centrosomes and regulate the dynamics and organization of microtubules. Human EB1 family proteins, which include EB1, EBF3, and RP1, also associate with the tumor suppressor protein adenomatous polyposis coli (APC) and p150glued, a component of the dynactin complex. The structural basis for interaction between human EB1 family proteins and their associated proteins has not been defined in detail. EB1 family proteins have a calponin homology (CH) domain at their N terminus and an EB1-like C-terminal motif at their C terminus; the functional importance of these domains has not been determined. To better understand functions of human EB1 family proteins and to reveal functional similarities and differences among these proteins, we performed detailed characterizations of interactions between human EB1 family proteins and their associated proteins. We show that amino acids 1-133 of EB1 and EBF3 and the corresponding region of RP1, which contain a CH domain, are necessary and sufficient for binding microtubules, thus demonstrating for the first time that a CH domain contributes to binding microtubules. EB1 family proteins use overlapping but different regions that contain the EB1-like C-terminal motif to associate with APC and p150glued. Neither APC nor p150glued binding domain is necessary for EB1 or EBF3 to induce microtubule bundling, which requires amino acids 1-181 and 1-185 of EB1 and EBF3, respectively. We also determined that the EB1 family protein-binding regions are amino acids 2781-2820 and 18-111 of APC and p150glued, respectively.     

Characterization of the functional domains of human foamy virus integrase using chimeric integrases

Retroviral integrases insert viral DNA into target DNA. In this process they recognize their own DNA specifically via functional domains. In order to analyze these functional domains, we constructed six chimeric integrases by swapping domains between HIV-1 and HFV integrases, and two point mutants of HFV integrase. Chimeric integrases with the central domain of HIV-1 integrase had strand transfer and disintegration activities, in agreement with the idea that the central domain determines viral DNA specificity and has catalytic activity. On the other hand, chimeric integrases with the central domain of HFV integrase did not have any enzymatic activity apart from FFH that had weak disintegration activity, suggesting that the central domain of HFV integrase was defective catalytically or structurally. However, these inactive chimeras were efficiently complemented by the point mutants (D164A and E200A) of HFV integrase, indicating that the central domain of HFV integrase possesses potential enzymatic activity but is not able to recognize viral or target DNA without the help of its homologous N-terminal and C-terminal domains.     

Characterization of functional domains in human tissue-type plasminogen activator with the use of monoclonal antibodies

Two murine monoclonal antibodies (MA-2G6 and MA-1C8), secreted by hybridomas obtained by fusion of myeloma cells with spleen cells from mice immunized with human tissue-type plasminogen activator (t-PA), inhibited the activity of t-PA on fibrin plates. MA-2G6 inhibited the amidolytic activity of t-PA and did not react with t-PA in which the active-site serine was blocked with diisopropylfluorophosphate nor with t-PA in which the active-site histidine was alkylated by reaction with D-Ile-Pro-Arg-CH2Cl. This indicated that MA-2G6 is directed against an epitope covering the active site of t-PA. MA-1C8 did not inhibit the amidolytic activity of t-PA, but abolished both the binding of t-PA to fibrin and the stimulatory effect of fibrin on the activation of plasminogen by t-PA. Thus MA-1C8 is directed against an epitope which covers the fibrin-binding site of t-PA. The A and B chains of partially reduced two-chain t-PA were separated by immunoadsorption on immobilized MA-1C8 and MA-2G6. The purified B chain reacted with MA-2G6 but not with MA-1C8 and activated plasminogen following Michaelis-Menten kinetics with kinetic constants similar to those of intact t-PA (Km = 100 microM and kcat = 0.02 s-1). However, fibrin or CNBr-digested fibrinogen did not stimulate the activation of plasminogen by the B chain. The purified A chain reacted with MA-1C8 but not with MA-2G6. It bound to fibrin with an affinity similar to that of intact t-PA but did not activate plasminogen. It is concluded that the active center of t-PA is located in the B chain and the fibrin-binding site in the A-chain. Both functional domains are required for the regulation by fibrin of the t-PA-mediated activation of plasminogen.     

Repeated phosphopeptide motifs in human Claspin are phosphorylated by Chk1 and mediate Claspin function

Claspin is a checkpoint protein involved in ATR (ataxia telangiectasia mutated- and Rad3-related)-dependent Chk1 activation in Xenopus and human cells. In Xenopus, Claspin interacts with Chk1 after DNA damage through a region containing two highly conserved repeats, which becomes phosphorylated during the checkpoint response. Because this region is also conserved in human Claspin, we investigated the regulation and function of these potential phosphorylation sites in human Claspin. We found that Claspin is phosphorylated in vivo at Thr-916 in response to replication stress and UV damage. Mutation of these phosphorylation sites on Claspin inhibited Claspin-Chk1 interaction in vivo, impaired Chk1 activation, and induced premature chromatin condensation in cells, indicating a defect in replication checkpoint. In addition, we found that Thr-916 on Claspin is phosphorylated by Chk1, suggesting that Chk1 regulates Claspin during checkpoint response. These results together indicate that phosphorylation of Claspin repeats in human Claspin is important for Claspin function and the regulation of Claspin-Chk1 interaction in human cells.     

Characterization of functional domains of the SMN protein in vivo

The Survival of Motor Neurons (SMN) is the disease gene of spinal muscular atrophy. We have previously established a genetic system based on the chicken pre-B cell line DT40, in which expression of SMN protein is regulated by tetracycline, to study the function of SMN in vivo. Depletion of SMN protein is lethal to these cells. Here we tested the functionality of mutant SMN proteins by determining their capacity to rescue the cells after depletion of wild-type SMN. Surprisingly, all of the spinal muscular atrophy-associated missense mutations tested were able to support cell viability and proliferation. Deletion of the amino acids encoded by exon 7 of the SMN gene resulted in a partial loss of function. A mutant SMN protein lacking both the tyrosine/glycine repeat (in exon 6) and exon 7 failed to sustain viability, indicating that the C terminus of the protein is critical for SMN activity. Interestingly, the Tudor domain of SMN, encoded by exon 3, does not appear to be essential for SMN function since a mutant deleted of this domain restored cell viability. Unexpectedly, a chicken SMN mutant (DeltaN39) lacking the N-terminal 39 amino acids that encompass the Gemin2-binding domain also rescued the lethal phenotype. Moreover, the level of Gemin2 in DeltaN39-rescued cells was significantly reduced, indicating that Gemin2 is not required for DeltaN39 to perform the essential function of SMN in DT40 cells. These findings suggest that SMN may perform a novel function in DT40 cells.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Characterization of the topology and functional domains of RKTG

RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61-71 and 299-303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.     

Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.     

Claspin promotes normal replication fork rates in human cells

The S phase-specific adaptor protein Claspin mediates the checkpoint response to replication stress by facilitating phosphorylation of Chk1 by ataxia-telangiectasia and Rad3-related (ATR). Evidence suggests that these components of the ATR pathway also play a critical role during physiological S phase. Chk1 is required for high rates of global replication fork progression, and Claspin interacts with the replication machinery and might therefore monitor normal DNA replication. Here, we have used DNA fiber labeling to investigate, for the first time, whether human Claspin is required for high rates of replication fork progression during normal S phase. We report that Claspin-depleted HeLa and HCT116 cells display levels of replication fork slowing similar to those observed in Chk1-depleted cells. This was also true in primary human 1BR3 fibroblasts, albeit to a lesser extent, suggesting that Claspin is a universal requirement for high replication fork rates in human cells. Interestingly, Claspin-depleted cells retained significant levels of Chk1 phosphorylation at both Ser317 and Ser345, raising the possibility that Claspin function during normal fork progression may extend beyond facilitating phosphorylation of either individual residue. Consistent with this possibility, depletion of Chk1 and Claspin together doubled the percentage of very slow forks, compared with depletion of either protein alone.     

Characterization of the functional domains of Escherichia coli RNase II

RNase II is a single-stranded-specific 3'-exoribonuclease that degrades RNA generating 5'-mononucleotides. This enzyme is the prototype of an ubiquitous family of enzymes that are crucial in RNA metabolism and share a similar domain organization. By sequence prediction, three different domains have been assigned to the Escherichia coli RNase II: two RNA-binding domains at each end of the protein (CSD and S1), and a central RNB catalytic domain. In this work we have performed a functional characterization of these domains in order to address their role in the activity of RNase II. We have constructed a large set of RNase II truncated proteins and compared them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants were determined using different single- or double-stranded substrates. The results obtained revealed that S1 is the most important domain in the establishment of stable RNA-protein complexes, and its elimination results in a drastic reduction on RNA-binding ability. In addition, we also demonstrate that the N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. Moreover, the biochemical results obtained with RNB mutant that lacks both putative RNA-binding domains, revealed the presence of an additional region involved in RNA binding. Such region, was identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain.     

Structural and functional domains in human tumour necrosis factors.

Human tumour necrosis factors (hTNFs) alpha and beta are related pleiotropic cytokines which share many activities and compete with each other for binding to two receptor components on many cell types. Although structural and biological data indicate that the active form of hTNF-alpha may be a symmetrical trimer, the manner in which hTNFs interact with their receptors to trigger a myriad of cell type-dependent responses is not clear. A combination of chemical modification, epitope mapping and site-directed mutagenesis approaches suggest that at least four distinct peptide sequences are important for the biological activity of hTNF-alpha. In particular, certain peptide sequences between amino acid positions 11 and 35 in hTNF-alpha appear to be critical for receptor binding and triggering biological responses. The recent cloning of the two hTNF-alpha/beta receptors opens the way for precise mapping of the functional domains in hTNFs.     

Characterization of the functional domains of galactosylceramide expression factor 1 in MDCK cells

We previously reported that GalCer expression factor 1 (GEF-1), a rat homologue of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), induced GalCer expression, morphological changes, and cell growth inhibition in COS-7 cells. In this study, we describe the characterization of GEF-1 in MDCK cells. Overexpression of GEF-1 in MDCK (MDCK/GEF-1) cells showed GalCer-derived sulfatide expression as well as dramatic morphological changes, but not cell growth suppression. The enzyme activity and the mRNA level of UDP-galactose:ceramide galactosyltransferase (CGT) increased significantly in MDCK/GEF-1 cells compared with control cells. GEF-1 molecule is composed of four domains; a zinc-finger (Z), a proline-rich (P), a coiled-coil (C), and a proline/glutamine-rich (Q) domain. MDCK cells transfected with various GEF-1 deletion mutants were examined for morphology and for glycolipid expression. MDCK cells transfected with Z-domain deletion mutant (MDCK/PCQ) and those with both Z- and P-domains deletion mutant (MDCK/CQ) were similar to those with a wild-type GEF-1 (MDCK/ZPCQ) in shape, exhibiting fibroblast-like cells, whereas those with the other deletion mutants showed no morphological changes, exhibiting typical epithelial-like cells. On the other hand, MDCK/ZPCQ, MDCK/PCQ, MDCK/CQ, and MDCK/Q cells expressed sulfatide, whereas those with the other deletion mutants that did not include the Q-domain showed neither GalCer nor sulfatide expression. Thus, the correlation between fibroblast-like cells in shape and the glycolipid expression was good in these deletion mutants except MDCK/Q cells, which showed epithelial-like cells, but expressed sulfatide. The glycolipid expression paralleled CGT mRNA levels. Taking these results together, it is suggested that only the Q-domain may be essential for the role of GEF-1 in inducing CGT mRNA, whereas the Q-domain together with the C-domain may be required for the induction of morphological changes in MDCK cells.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

Characterization of the glucagon receptor and its functional domains using monoclonal antibodies

Four monoclonal antibodies, designated 4H11, 6E10, 2C5, and 3E9 were prepared against partially purified rat hepatic glucagon receptor. These antibodies were characterized by their ability to recognize the glucagon receptor in target tissues using immunoblot and immunoprecipitation procedures. The antibodies recognized a 62-kDa receptor band in rat liver, kidney, and adipose tissue but not in lung, adrenals, and erythrocytes, indicating a high degree of specificity. These antibodies recognize different antigenic determinants; the 6E10 and 2C5 bind protein epitopes, while 4H11 and 3E9 bind carbohydrate epitopes. Furthermore, proteolytic mapping of the glucagon receptor established that monoclonal antibodies 6E10 and 2C5 recognize different domains of the receptor molecule. These antibodies were used to study the immunochemical similarities among the receptors from different species and to assess the topological location of the ligand-binding site. By combining the techniques of affinity cross-linking, proteolytic mapping, and antibody binding, we have identified the location of the glucagon-binding site near to the COOH-terminal domain of the receptor.     

Roles of replication fork-interacting and Chk1-activating domains from Claspin in a DNA replication checkpoint response

Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265-605) that associates with Cdc45, DNA polymerase epsilon, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265-331 and 470-600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776-905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1.     

The functional domains of human ventricular myosin light chain 1

The biological functions of the myosin light chain 1 (LC1) have not been clearly elucidated yet. In this work we cloned and expressed N- and C- terminal fragments of human ventricular LC1 (HVLC1) containing amino acid residues 1-98 and 99-195 and two parts, NN and NC of N fragment in GST-fusion forms, respectively. Using GST pull-down assay, the direct binding experiments of LC1 with rat cardiac G-actin, F-actin and thin filaments, as well as rat cardiac myosin heavy chain (RCMHC) have been performed. Furthermore, the recombinant complexes of rat myosin S1 with N- and C-fragments, as well as the whole molecular of HVLC1 were generated. The results suggested that both binding sites of HVLC1 for actin and myosin heavy chain are positioned in its N-terminal fragment, which may contain several actin-binding sites in tandem. The polymerization of G-actin, the tropomyosin and troponin molecules located in the thin filaments do not hinder the binding of N-terminal fragment of HVLC1 with actin and thin filaments in vitro. The recombinant complex of rat cardiac myosin S1 (RCMS1) with N fragment of HVLC1 greatly decreased actin-activated Mg(2+)-ATPase activity for lack of C fragment. We conclude that the N-fragment is the binding domain of human ventricular LC1, whereas the C-fragment serves as a functional domain, which may be more involved in the modulation of the actin-activated ATPase activity of myosin.     

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.     

Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an α-β hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V_max than the full-length form without changes in K_m, which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic α-helix (bold) within amino acid residues 10–24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic α-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.