Insights into cargo sorting by SNX32 and its role in neurite outgrowth.

Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Here, we established that SNX32, an SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member associates with SNX4 via its BAR domain and the residues A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 that lie at the interface of these two SNX proteins mediate this association. SNX32, via its PX domain, interacts with the transferrin receptor (TfR) and Cation-Independent Mannose-6-Phosphate Receptor (CIMPR), and the conserved F131 in its PX domain is important in stabilizing these interactions. Silencing of SNX32 leads to a defect in intracellular trafficking of TfR and CIMPR. Further, using SILAC-based differential proteomics of the wild-type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin superfamily member, as a potential interactor of SNX32 in SHSY5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In neuroglial cell lines, silencing of SNX32 leads to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32-depleted cells led us to propose that SNX32 may contribute to maintaining the neuroglial coordination via its role in BSG trafficking and the associated monocarboxylate transporter activity. Taken together, our study showed that SNX32 mediates the trafficking of specific cargo molecules along distinct pathways.

Monitoring Endosomal Cargo Retrieval to the Trans-Golgi Network by Microscopic and Biochemical Approaches.

The endosomal recycling pathway plays a crucial role in diverse physiologically important biological processes such as cell-to-cell signaling, nutrient uptake, immune response, and autophagy. A selective subset of these recycling cargoes, mostly transmembrane proteins, is retrieved from endosomes to the trans-Golgi network (TGN) by a retrograde transport process. Endosome-to-TGN retrograde trafficking is crucial for maintaining cellular homeostasis and signaling by preventing proteins and lipids from degradation in the lysosome. Many of the membrane sorting machinery, such as the retromer complex and sorting nexins (SNXs) are involved in endosomal retrieval and recycling of various transmembrane proteins. Recent technological advances in the resolution of light microscopy and unbiased analytical approaches in quantitative image analysis enable us to explore and understand the regulation of membrane trafficking pathways in greater detail. In this chapter, we describe quantitative imaging-based methods for analyzing the roles of proteins involved in the retrograde trafficking in retromer dependent or independent fashion, using cation-independent mannose-6-phosphate receptor (CIM6PR) as an example.

Measuring Plasma Membrane Recycling Using Microscopic and Biochemical Approaches.

The endocytic pathway has an intricate network of vesicular compartments carrying a variety of proteins referred to as cargoes. Endosomal trafficking is exclusively required to transport these cargoes through various intracellular routes for their delivery to the site of action. Among these, recycling of cargoes to the plasma membrane is a crucial pathway for the efficient functioning of the cell. Hence, endosomal cargo recycling assays are crucial to gain insight into the molecular mechanism governing recycling of the cargoes and in turn to understand their key role in maintaining cellular physiology. These assays have been efficiently utilized to study the recycling of adhesion molecules, transporters, channels, receptors, and so on to the plasma membrane. The basic methodology involves labelling of the cargo at the surface, allowing its internalization followed by direct or indirect measurement of the amount of the cargo recycled back to the plasma membrane. These microscopy-based and biochemical methods can be used as a tool to study the role of various trafficking or signaling molecules on the cell surface involved with the recycling of the membrane proteins, by altering their expression either by silencing or overexpressing the gene.

Syntaxin 7 contributes to breast cancer cell invasion by promoting invadopodia formation.

Invasion in various cancer cells requires coordinated delivery of signaling proteins, adhesion proteins, actin-remodeling proteins and proteases to matrix-degrading structures called invadopodia. Vesicular trafficking involving SNAREs plays a crucial role in the delivery of cargo to the target membrane. Screening of 13 SNAREs from the endocytic and recycling route using a gene silencing approach coupled with functional assays identified syntaxin 7 (STX7) as an important player in MDA-MB-231 cell invasion. Total internal reflection fluorescence microscopy (TIRF-M) studies revealed that STX7 resides near invadopodia and co-traffics with MT1-MMP (also known as MMP14), indicating a possible role for this SNARE in protease trafficking. STX7 depletion reduced the number of invadopodia and their associated degradative activity. Immunoprecipitation studies revealed that STX7 forms distinct SNARE complexes with VAMP2, VAMP3, VAMP7, STX4 and SNAP23. Depletion of VAMP2, VAMP3 or STX4 abrogated invadopodia formation, phenocopying what was seen upon lack of STX7. Whereas depletion of STX4 reduced MT1-MMP level at the cell surfaces, STX7 silencing significantly reduced the invadopodia-associated MT1-MMP pool and increased the non-invadosomal pool. This study highlights STX7 as a major contributor towards the invadopodia formation during cancer cell invasion. This article has an associated First Person interview with the first author of the paper.

PI Kinase-EhGEF2-EhRho5 axis contributes to LPA stimulated macropinocytosis in Entamoeba histolytica.

Entamoeba histolytica is a protozoan responsible for several pathologies in humans. Trophozoites breach the intestinal site to enter the bloodstream and thus traverse to a secondary site. Macropinocytosis and phagocytosis, collectively accounting for heterophagy, are the two major processes responsible for sustenance of Entamoeba histolytica within the host. Both of these processes require significant rearrangements in the structure to entrap the target. Rho GTPases play an indispensable role in mustering proteins that regulate cytoskeletal remodelling. Unlike phagocytosis which has been studied in extensive detail, information on machinery of macropinocytosis in E. histolytica is still limited. In the current study, using site directed mutagenesis and RNAi based silencing, coupled with functional studies, we have demonstrated the involvement of EhRho5 in constitutive and LPA stimulated macropinocytosis. We also report that LPA, a bioactive phospholipid present in the bloodstream of the host, activates EhRho5 and translocates it from cytosol to plasma membrane and endomembrane compartments. Using biochemical and FRAP studies, we established that a PI Kinase acts upstream of EhRho5 in LPA mediated signalling. We further identified EhGEF2 as a guanine nucleotide exchange factor of EhRho5. In the amoebic trophozoites, EhGEF2 depletion leads to reduced macropinocytic efficiency of trophozoites, thus phenocopying its substrate. Upon LPA stimulation, EhGEF2 is found to sequester near the plasma membrane in a wortmannin sensitive fashion, explaining a possible mode for activation of EhRho5 in the amoebic trophozoites. Collectively, we propose that LPA stimulated macropinocytosis in E. histolytica is driven by the PI Kinase-EhGEF2-EhRho5 axis.

EhRho6-mediated actin degradation in Entamoeba histolytica is associated with compromised pathogenicity.

Entamoeba histolytica causes amoebiasis which is a major health concern in developing countries. E. histolytica pathogenicity has been implicated to a large repertoire of small GTPases which switch between the inactive GDP bound state and the active GTP bound state with the help of guanine nucleotide exchange factors (GEFs) and GTPase activating protein (GAPs). Rho family of small GTPases are well known to modulate the actin cytoskeletal dynamics which plays a major role in E. histolytica pathogenicity. Here, we report an atypical amoebic RhoGEF, and its preferred substrate EhRho6, which, upon overexpression abrogated the pathogenic behavior of the amoeba such as adhesion to host cell, monolayer destruction, erythrophagocytosis, and formation of actin dots. A causative immunoblot analysis revealed actin degradation in the EhRho6 overexpressing trophozoites that could be inhibited by blocking the amoebic proteasomal pathway. A careful analysis of the results from a previously published transcriptomics study, in conjunction with our observations, led to the identification of a clade of Rho GTPases in this pathogenic amoeba which we hypothesize to have implications during the amoebic encystation.

Binding with heat shock cognate protein HSC70 fine-tunes the Golgi association of the small GTPase ARL5B.

ARL5B, an ARF-like small GTPase localized to the trans-Golgi, is known for regulating endosome-Golgi trafficking and promoting the migration and invasion of breast cancer cells. Although a few interacting partners have been identified, the mechanism of the shuttling of ARL5B between the Golgi membrane and the cytosol is still obscure. Here, using GFP-binding protein (GBP) pull-down followed by mass spectrometry, we identified heat shock cognate protein (HSC70) as an additional interacting partner of ARL5B. Our pull-down and isothermal titration calorimetry (ITC)-based studies suggested that HSC70 binds to ARL5B in an ADP-dependent manner. Additionally, we showed that the N-terminal helix and the nucleotide status of ARL5B contribute to its recognition by HSC70. The confocal microscopy and cell fractionation studies in MDA-MB-231 breast cancer cells revealed that the depletion of HSC70 reduces the localization of ARL5B to the Golgi. Using in vitro reconstitution approach, we provide evidence that HSC70 fine-tunes the association of ARL5B with Golgi membrane. Finally, we demonstrated that the interaction between ARL5B and HSC70 is important for the localization of cation independent mannose-6-phosphate receptor (CIMPR) at Golgi. Collectively, we propose a mechanism by which HSC70, a constitutively expressed chaperone, modulates the Golgi association of ARL5B, which in turn has implications for the Golgi-associated functions of this GTPase.

PLP2 drives collective cell migration via ZO-1-mediated cytoskeletal remodeling at the leading edge in human colorectal cancer cells.

Collective cell migration (CCM), in which cell-cell integrity remains preserved during movement, plays an important role in the progression of cancer. However, studies describing CCM in cancer progression are majorly focused on the effects of extracellular tissue components on moving cell plasticity. The molecular and cellular mechanisms of CCM during cancer progression remain poorly explored. Here, we report that proteolipid protein 2 (PLP2), a colonic epithelium-enriched transmembrane protein, plays a vital role in the CCM of invasive human colorectal cancer (CRC) epithelium by modulating leading-edge cell dynamics in 2D. The extracellular pool of PLP2, secreted via exosomes, was also found to contribute to the event. During CCM, the protein was found to exist in association with ZO-1 (also known as TJP1) and to be involved in the positioning of the latter at the migrating edge. PLP2-mediated positioning of ZO-1 at the leading edge further alters actin cytoskeletal organization that involves Rac1 activation. Taken together, our findings demonstrate that PLP2, via its association with ZO-1, drives CCM in CRC epithelium by modulating the leading-edge actin cytoskeleton, thereby opening up new avenues of cancer research. This article has an associated First Person interview with the first author of the paper.

Protein-templated gold nanoclusters as specific bio-imaging probes for the detection of Hg(ii) ions in in vivo and in vitro systems: discriminating between MDA-MB-231 and MCF10A cells.

Gold nanoclusters (AuNCs) synthesized within a protein (Human Serum Albumin, HSA) template exhibited intense red luminescence accompanied by a quantum yield >10% and remarkable photo and cluster-core stability for a prolonged period (more than a year). These photoluminescent nanoclusters (NCs) were resistant to chemical and thermal perturbations but break down selectively and highly sensitively in the presence of mercury, Hg(ii), ions. The AuNCs were efficient in quantifying Hg(ii) ions in solution as well as bound to the hormone insulin. By exploiting the auto-fluorescence of these AuNCs, we demonstrated that our AuNCs were able to sense Hg(ii) ions at single-molecule sensitivity using Fluorescence Correlation Spectroscopy (FCS), highlighting a detection limit in the sub-nanomolar regime. The translational diffusion time of the AuNCs decreased significantly upon the interaction with Hg(ii) ions and resulted in the formation of smaller sized clusters. A cell viability study reveals the non-toxic nature of these nano-probes, which thus can be used for cell imaging. Interestingly, a cell line-based study reveals that the fluorescence intensity of AuNCs could be detected in cancerous MDA-MB-231 cells but not in non-cancerous breast-derived MCF10A cells. Further, time lapse fixed cell imaging by confocal microscopy revealed that the fluorescence of AuNCs could be quenched by Hg(ii) ions inside the MDA-MB-231 cells. Thus the objective of our study is to appraise the sensitive in vivo as well as in vitro detection of Hg(ii) ions using AuNCs as a probe.

EhC2B, a C2 domain-containing protein, promotes erythrophagocytosis in Entamoeba histolytica via actin nucleation.

Remodelling of the actin cytoskeleton in response to external stimuli is obligatory for many cellular processes in the amoebic cell. A rapid and local rearrangement of the actin cytoskeleton is required for the development of the cellular protrusions during phagocytosis, trogocytosis, migration, and invasion. Here, we demonstrated that EhC2B, a C2 domain-containing protein, is an actin modulator. EhC2B was first identified as an effector of EhRab21 from E. histolytica. In vitro interaction studies including GST pull-down, fluorescence-based assay and ITC also corroborated with our observation. In the amoebic trophozoites, EhC2B accumulates at the pseudopods and the tips of phagocytic cups. FRAP based studies confirmed the recruitment and dynamics of EhC2B at the phagocytic cup. Moreover, we have shown the role of EhC2B in erythrophagocytosis. It is well known that calcium-dependent signal transduction is essential for the cytoskeletal dynamics during phagocytosis in the amoebic parasite. Using liposome pelleting assay, we demonstrated that EhC2B preferentially binds to the phosphatidylserine in the presence of calcium. The EhC2B mutants defective in calcium or lipid-binding failed to localise beneath the plasma membrane. The cells overexpressing these mutants have also shown a significant reduction in erythrophagocytosis. The role of EhC2B in erythrophagocytosis and pseudopod formation was also validated by siRNA-based gene knockdown approach. Finally, with the help of in vitro nucleation assay using fluorescence spectroscopy and total internal reflection fluorescence microscopy, we have established that EhC2B is an actin nucleator. Collectively, based on the results from the study, we propose that EhC2B acts like a molecular bridge which promotes membrane deformation via its actin nucleation activity during the progression of the phagocytic cup in a calcium-dependent manner.

Rab GTPases take centre stage in understanding Entamoeba histolytica biology.

Rab GTPases constitute the largest subgroup in the Ras superfamily of GTPases. It is well established that different Rab GTPases are localized in discrete subcellular localization and regulate the membrane trafficking in nearly all eukaryotic cells. Rab GTPase diversity is often regarded as an expression of vesicular trafficking complexity. The pathogenic amoeba Entamoeba histolytica harbours 91 Rab GTPases which is the highest among the currently available genome sequences from the eukaryotic kingdom. Here, we review the current status of amoebic Rab GTPases diversity, unique biochemical and structural features and summarise their predicted regulators. We discuss how amoebic Rab GTPases are involved in cellular processes such as endocytosis, phagocytosis, and invasion of host cellular components, which are essential for parasite survival and virulence.

SNX27-retromer assembly recycles MT1-MMP to invadopodia and promotes breast cancer metastasis.

A variety of metastatic cancer cells use actin-rich membrane protrusions, known as invadopodia, for efficient ECM degradation, which involves trafficking of proteases from intracellular compartments to these structures. Here, we demonstrate that in the metastatic breast cancer cell line MDA-MB-231, retromer regulates the matrix invasion activity by recycling matrix metalloprotease, MT1-MMP. We further found that MT2-MMP, another abundantly expressed metalloprotease, is also invadopodia associated. MT1- and MT2-MMP showed a high degree of colocalization but were located on the distinct endosomal domains. Retromer and its associated sorting nexin, SNX27, phenocopied each other in matrix degradation via selectively recycling MT1-MMP but not MT2-MMP. ITC-based studies revealed that both SNX27 and retromer could directly interact with MT1-MMP. Analysis from a publicly available database showed SNX27 to be overexpressed or frequently altered in the patients having invasive breast cancer. In xenograft-based studies, SNX27-depleted cell lines showed prolonged survival of SCID mice, suggesting a possible implication for overexpression of the sorting nexin in tumor samples.

Heavy subunit of cell surface Gal/GalNAc lectin (Hgl) undergoes degradation via endo-lysosomal compartments in Entamoeba histolytica.

The human gut parasite Entamoeba histolytica uses a multifunctional virulence factor, Hgl, a cell surface transmembrane receptor subunit of Gal/GalNAc lectin that contributes to adhesion, invasion, cytotoxicity and immune response in the host. At present, the physiologic importance of Hgl receptor is mostly known for pathogenicity of E. histolytica. However, the molecular mechanisms of Hgl trafficking events and their association with the intracellular membrane transport machinery are largely unknown. We used biochemical and microscopy-based assays to understand the Hgl trafficking in the amoebic trophozoites. Our results suggest that the Hgl is constitutively degraded through delivery into amoebic lysosome-like compartments. Further, we also observed that the Hgl was significantly colocalized with amoebic Rab GTPases such as EhRab5, EhRab7A, and EhRab11B. While, we detected association of Hgl with all these Rab GTPases in early vacuolar compartments, only EhRab7A remains associated with Hgl till its transport to amoebic lysosome-like compartments.

Atypical Switch-I Arginine plays a catalytic role in GTP hydrolysis by Rab21 from Entamoeba histolytica.

Entamoeba histolytica, the causative agent of amoebic dysentery, liver abscess and colitis, exploits its vesicular trafficking machinery for survival and virulence. Rab family of small GTPases play a key role in the vesicular transport by undergoing the GTP/GDP cycle which is central to the biological processes. Amoebic genome encodes several atypical Rab GTPases which are unique due to absence of conserved sequence motif(s) or atypical residues in their catalytic site [Saito-Nakano et al., 2005 ]. Previously, EhRab21 has been reported to involve in amoebic invasion and migration [Emmanuel et al., 2015 ]. The conserved Glutamine of switch-II region is universally accepted to be crucial for GTP hydrolysis. Mutations that reduce the sidechain polarity of Glutamine render the protein GTPase activity deficient [Krengel et al., 1990]. Here, we report a catalytic role of atypical switch-I Arginine (R36) in intrinsic GTP hydrolysis catalysed by EhRab21. Unlike the GTPase activity deficient QL mutants, the GTPase activity of EhRab21Q64L was found to be marginally enhanced compared to the wild-type protein. Although EhRab21R36L mutant showed normal GTPase activity, the double mutant (R36L/Q64L) was found to be GTPase deficient. Thus, EhRab21 is a unique member of small GTPase family in which an atypical switch-I Arginine is capable of driving GTP hydrolysis independent of the conserved switch-II Glutamine.

Structural and thermodynamic characterization of metal binding in Vps29 from Entamoeba histolytica: implication in retromer function.

Vps29 is the smallest subunit of retromer complex with metallo-phosphatase fold. Although the role of metal in Vps29 is in quest, its metal binding mutants has been reported to affect the localization of the retromer complex in human cells. In this study, we report the structural and thermodynamic consequences of these mutations in Vps29 from the protozoan parasite, Entamoeba histolytica (EhVps29). EhVps29 is a zinc binding protein as revealed by X-ray crystallography and isothermal titration calorimetry. The metal binding pocket of EhVps29 exhibits marked differences in its 3-dimensional architecture and metal coordination in comparison to its human homologs and other metallo-phosphatases. Alanine substitutions of the metal-coordinating residues showed significant alteration in the binding affinity of EhVps29 for zinc. We also determined the crystal structures of metal binding defective mutants (D62A and D62A/H86A) of EhVps29. Based on our results, we propose that the metal atoms or the bound water molecules in the metal binding site are important for maintaining the structural integrity of the protein. Further cellular studies in the amoebic trophozoites showed that the overexpression of wild type EhVps29 leads to reduction in intracellular cysteine protease activity suggesting its crucial role in secretion of the proteases.

Essential and selective role of SNX12 in transport of endocytic and retrograde cargo.

The endosomal protein-sorting machineries play vital roles in diverse physiologically important cellular processes. Much of the core membrane-sorting apparatus is conserved in evolution, such as retromer, which is involved in the recycling of a diverse set of cargoes via the retrograde trafficking route. Here, in an RNAi-based loss-of-function study, we identified that suppression of SNX12 leads to a severe blockage in CIM6PR (also known as IGF2R) transport and alters the morphology of the endocytic compartments. We demonstrate that SNX12 is involved in the early phase of CIM6PR transport, and mediates receptor recycling upstream of the other well-established SNX components of retromer. Ultra-structural analysis revealed that SNX12 resides on tubulo-vesicular structures, despite it lacking a BAR domain. Furthermore, we illustrate that SNX12 plays a key role in intraluminal vesicle formation and in the maturation of a subpopulation of early endosomes into late endosomes, thereby regulating selective endocytic transport of cargo for degradation. This study therefore provides evidence for the existence of early endosomal subpopulations that have differential roles in the sorting of the cargoes along endocytic degradative pathways.

The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite.

Role of EhRab7A in phagocytosis of type 1 fimbriated E. coli by Entamoeba histolytica.

Entamoeba histolytica, the causative agent of amoebic colitis and liver abscess in human, ingests the intestinal bacteria and variety of host cells. Phagocytosis of bacteria by the amebic trophozoite has been reported to be important for the virulence of the parasite. Here, we set out to characterize different stages of phagocytosis of type 1 E. coli and investigated the role of a set of amoebic Rab GTPases in the process. The localizations of the Rab GTPases during different stages of the phagocytosis were investigated using laser scanning confocal microscopy and their functional relevance were determined using fluorescence activated cell sorter based assay as well as colony forming unit assay. Our results demonstrate that EhRab7A is localized on the phagosomes and involved in both early and late stages of type 1 E. coli phagocytosis. We further showed that the E. coli or RBC containing phagosomes are distinct from the large endocytic vacuoles in the parasite which are exclusively used to transport human holotransferrin and low density lipoprotein. Remarkably, type 1 E. coli uptake was found to be insensitive to cytochalasin D treatment, suggesting that the initial stage of E. coli phagocytosis is independent of the formation of actin filaments.

Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3.

The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding.

Deciphering the role of Atg5 in nucleotide dependent interaction of Rab33B with the dimeric complex, Atg5-Atg16L1.

Autophagy is a lysosomal degradation pathway that degrades cytosolic constituents, including whole organelles and intracellular pathogens. Previous studies on various autophagy related genes revealed the importance of the Atg12-Atg5-Atg16 complex in autophagy. Atg16L1 is an effector of Golgi-resident Rab33B and the molecular mechanism of the interaction of Rab33B with either Atg16L1 or in complex with Atg5 is still elusive. In the current study, using the pull down and calorimetric approaches, we have dissected the molecular insights into the interaction of Rab33B with different regions of mouse Atg16L1 as well as with the dimeric complex, Atg5-mAtg16L1. Our in vitro observation suggests that Atg5 is pre-requisite for the augmented nucleotide dependent interaction of Rab33B with the dimeric complex, Atg5-Atg16L1. Moreover, the results reported here suggest that Arg-24 of Atg16L1 is crucial for its interaction with Atg5 which will have further implication in the binding of the dimeric complex to Rab33B.