Synaptic vesicle (SV) recycling in the presynapse is essential for the maintenance of neurotransmission. During mild stimulation clathrin-mediated endocytosis (CME) dominates, however during intense stimulation activity-dependent bulk endocytosis (ADBE) is the dominant form of membrane retrieval. The aim of this thesis was to determine how the signalling molecule GSK3 controlled ADBE, with the hypothesis that this enzyme was required at multiple stages of this endocytosis mode. I also hoped to identify a specific cargo for ADBE. I found that during intense action potential stimulation, a localised calcium increase is necessary for the activation of Akt, which inhibited GSK3. This activation was mediated via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Furthermore, I found that phosphatidylinositol 4-kinaseIIα (PI4KIIα), a molecule whose abundance is regulated by GSK3, had a key role in ADBE. Specifically, I found that the absence of PI4KIIα accelerated CME but inhibited ADBE and that PI4KIIα controls CME and ADBE via distinct mechanisms. The PI4KIIα study revealed potential cross-talk between CME and ADBE. To determine whether modulation of either endocytosis mode impacts on the other, the retrieval of genetically-encoded reporters of SV cargo was monitored during intense stimulation during inhibition of either CME or ADBE. The recovery of almost all SV cargo was unaffected by ADBE inhibition but was arrested by abolishing CME. In contrast, VAMP4-pHluorin retrieval was perturbed by inhibiting ADBE and not by blocking CME. Knockdown of VAMP4 also arrested ADBE, indicating that in addition to being the first identified ADBE cargo, it is also essential for this endocytosis mode to proceed.

Endocytosis is important for the regulation of signal transduction and for the movement of essential cellular components from outside the cell to their appropriate intracellular compartment(s). Two established mechanisms of endocytosis are clathrinmediated (CME) and clathrin-independent endocytosis, and they are responsible for internalization of different ligands. In this study, the newly established technique of total internal reflection fluorescent microscopy (TIRF-M) was used, along with standard biochemical and molecular biological tools, to systematically study the sorting and early trafficking of two established ligands of endocytosis, transferrin (Tf) and epidermal growth factor (EGF). TIRF-M studies revealed that Tf binds its receptor that is located in large clathrin arrays positioned just below the surface of the cell and that these large clathrin platforms serves as the major site of CME at the plasma membrane. EGF endocytosis is very different and occurs as follows 1) the liganded EGFR recruits Rab5 to the plasma membrane, 2) Rab5 concentrates around vesicles containing liganded EGFR and 3) these vesicles co-localize with EEA1 enriched endosomes. EEA1 was shown to play a pivotal role in EGF endocytosis, establishing a new role for EEA1 in vesicle trafficking in addition to its role in tethering and fusion. Finally, WDFY2, a new FYVE domain protein was shown to decorate a specific subset of vesicles, upstream of the EEA1 vesicle pool that appear to participate in Tf endocytosis. These studies establish new functions and components of endocytosis that enhances our understanding of this complex process.

A hidatidose cística é uma zoonose causada pelo estágio larval (cisto hidático) do Echinococcus granulosus. O cisto hidático desenvolve-se nas vísceras dos hospedeiros intermediários como uma estrutura unilocular preenchida pelo líquido hidático, que contém os produtos de excreção/secreção do parasito. O antígeno B (AgB) de E. granulosus, um dos principais componentes antigênicos do líquido hidático, é uma lipoproteína oligomérica composta por subunidades de 8 kDa. Em solução o AgB existe como distintos oligômeros e agregados em equilíbrio, contudo, os oligômeros de aproximadamente 160 kDa são predominantes. A função do AgB não é totalmente clara, mas dois papeis principais são sugeridos: de molécula imunomoduladora e de carreador de lipídios. Neste estudo, nós investigamos se o AgB é capaz de interagir com diferentes tipos celulares e afetar sua viabilidade. O AgB foi purificado por cromatografia de imunoafinidade a partir de líquido hidático coletado de cistos hidáticos individuais. A internalização do AgB pelas linhagens A549 (célula epitelial de pulmão), NIH/3T3 (fibroblasto), RH (hepatócito) e J774 (macrófago) foi avaliada por imunofluorescência. A viabilidade celular foi analisada pelo ensaio de redução do MTT após incubação com AgB por 24 h. As quatro linhagens celulares foram capazes de internalizar o AgB a 37°C. Para avaliar o envolvimento de endocitose na internalização, células RH foram incubadas com AgB a 4°C, condição que inibe processos de endocitose. Nessa condição, o AgB não foi observado no citoplasma celular, sugerindo que vias de endocitose poderiam estar envolvidas. Células RH e A549 mostraram maior sensibilidade no ensaio de MTT. A taxa de redução de MTT no tratamento com 100 μg/ml de AgB foi de 68% e 57% para RH e A549, respectivamente. Nas mesmas condições, células NIH/3T3 e J774 mostraram respostas similares, o nível de redução do MTT foi 80% e 78%, respectivamente. A sensibilidade observada para as células A549 e RH poderia estar relacionada ao organotropismo por pulmões e fígado reportado na hidatidose cística. A internalização pelas células pode ser uma etapa necessária para a função do AgB como carreador de lipídios ou molécula imunomoduladora e durante esse processo, o AgB poderia causar dano celular nos tecidos do hospedeiro. / Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid cyst) of Echinococcus granulosus. The hydatid cyst develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. E. granulosus antigen B (AgB), one of the major antigenic component of hydatid fluid, is an oligomeric lipoprotein composed by small subunits with approximately 8 kDa. In solution, AgB exists as distinct oligomers and aggegates in equilibrium, however oligomers with approximately 160 kDa are prevalent. The AgB function is not completely clear, but two major roles are suggested: as an immunomodulatory molecule and as a lipid carrier. In this study, the ability of AgB to interact with different cell types and to affect cell viability was investigated. AgB was purified by immunoaffinity chromatography from hydatid fluid collected from individual hydatid cysts. The internalization of AgB by A549 (lung epithelial cell), NIH/3T3 (fibroblast), RH (hepatocyte) and J774 (macrophage) cells was evaluated by immunofluorescence. Cell viability was analyzed by MTT reduction assay after incubation with AgB by 24 h. The four cell lines tested were able to internalize AgB at 37°C. In order to evaluate the role of endocytosis in the internalization, RH cells were incubated with AgB at 4°C, condition that inhibits endocytosis processes. In this condition, AgB was not visualized on cell cytoplasm, suggesting that endocytosis pathways could be envolved. RH and A549 cells showed higher sensitivity on MTT assay. The MTT reduction level on the 100 μg/ml AgB treatment was 68% and 57% for RH and A549, respectively. In the same condition, NIH/3T3 e J774 cells showed similar responses, the MTT reduction level was 80% and 78%, respectively. The observed sensitivity on A549 and RH cells could be related to the organothropism by lungs and liver reported on cystic hydatid disease. The internalization by cells might be a necessary step to the AgB function as a lipid carrier or an immunomodulatory molecule and during this process, AgB could cause cell damage to the host tissues.

Magister Scientiae - MSc (Medical BioSciences) / Central nervous system (CNS) infections are a therapeutic challenge. This is partly due to insufficient drug penetration across the blood-brain barrier (BBB). The BBB is a specialized, highly selective, metabolically active physiological barrier that regulates the movement of molecules into-and-out of the brain. As a result, large hydrophilic antibiotics such as colistin poorly penetrate to the CNS. Colistin is an old 'last line of defence'; a gram-negative antibiotic that has seen its clinical re-emergence due to the surge of multidrug resistance (MDR) infections. However, owing to systemic toxicity, increasing the intravenous dosage, in order to obtain higher CNS penetration, is inimical. Chitosan (CS) based nanoparticles (NPs) have been proposed as drug delivery systems across the BBB. CS is a cationic, natural polysaccharide that has the ability to be complexed with multivalent polymers like dextran (DS) thus forming CS-DS NPs. Naturally, CS has remarkable inherent features such as biocompatibility, biodegradability, ability to encapsulate poorly soluble drugs and it is favourable for endothelial cell uptake. However, polymeric NPs (even those derived from natural polysaccharides) have limited use due to toxicity. Considering the vital role of the BBB, toxicity would denote dire effects on CNS functioning. Therefore, treatment of CNS infections fringes on a deeper understanding of the interactions between drug delivery systems and the BBB.

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBPα. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

La surface naturelle des prothèses, tels que des implants métalliques, n'est pas idéale pour l'obtention d'une bonne ostéo-intégration. Par conséquent, l'amélioration des propriétés de surface pour les rendre ostéo-condutrices ou ostéo-inductrices est souhaitable. La délivrance contrôlée de protéines ostéo-inductrices de la famille des bone morphogenetic proteins (BMPs) par la surface des matériaux implantables permettrait une formation osseuse optimisée et plus rapide autour de l'implant. En particulier, la BMP-2 est importante dans la phase initiale de la différenciation vers l'os. En raison de leur similarité avec les tissus naturels, l'utilisation des revêtements de biopolymères qui ont une bonne affinité avec les molécules bioactives, semble prometteuse pour le chargement et la délivrance de BMP-2. L'équipe a déjà mis au point un film à base des biopolymères hyaluronane et de poly(L-lysine), en utilisant la technique d'assemblage couche par couche. Ce film constitue un réservoir qui permet de présenter la BMP-2 "liée à la matrice". La bioactivité in vitro et les propriétés ostéo-inductrices in vivo de ces films ont déjà prouvées. Dans ce travail, nous avons cherché à mieux comprendre l'interaction de la BMP-2 avec le film et l'interaction des cellules avec la BMP-2. Tout d'abord, nous avons étudié la structure de la BMP-2 piégée dans les films et l'avons comparé à celle en solution; puis nous avons évalué l'impact du séchage, du stockage à long terme et de la stérilisation sur la structure du film et sa bioactivité. Enfin, nous avons étudié l'internalisation de la BMP-2 par les cellules en fonction de la réticulation du film et avons étudié la relation entre internalisation et voies de signalisation. / The natural surface of bulk prostheses materials, such as metallic implants, is not suitable for successful osteointegration of implants. Therefore, improving the surface to render it osteoconductive and osteoinductive is needed. The controlled delivery of osteoinductive bone morphogenetic proteins (BMPs) from the surface of implantable materials would enable faster and better bone formation around the implant. In particular, BMP-2 plays an important role in the early phase of differentiation of stems cells in bone cells. The coating of natural polymers that have a high affinity for BMP-2 would enable BMP retention and localized delivery at the implant surface. Using the layer-by-layer technique, we have developed a coating made of the biopolymers hyaluronan and poly(L-Lysine), which acts as a reservoir to trap BMP-2 and to present them to cells in a "matrix-bound" manner. The in vitro bioactivity and in vivo osteoinductive properties of BMP-2-loaded films have previously been proved. In this work, the aim is to further understand the interaction of the BMP-2 with the film and the uptake of BMP-2 by the cells. First, the secondary structure of matrix-bound BMP-2 was studied and compared to its structure in solution. Second, the impact of drying, long term storage and sterilization on film structure and bioactivity were assessed. Finally, we investigated if and how matrix-bound BMP-2 is internalized by the cells from the different cross-linked films, the internalization route and its relation to BMP-2 signaling.

A hidatidose cística é uma zoonose causada pelo estágio larval (cisto hidático) do Echinococcus granulosus. O cisto hidático desenvolve-se nas vísceras dos hospedeiros intermediários como uma estrutura unilocular preenchida pelo líquido hidático, que contém os produtos de excreção/secreção do parasito. O antígeno B (AgB) de E. granulosus, um dos principais componentes antigênicos do líquido hidático, é uma lipoproteína oligomérica composta por subunidades de 8 kDa. Em solução o AgB existe como distintos oligômeros e agregados em equilíbrio, contudo, os oligômeros de aproximadamente 160 kDa são predominantes. A função do AgB não é totalmente clara, mas dois papeis principais são sugeridos: de molécula imunomoduladora e de carreador de lipídios. Neste estudo, nós investigamos se o AgB é capaz de interagir com diferentes tipos celulares e afetar sua viabilidade. O AgB foi purificado por cromatografia de imunoafinidade a partir de líquido hidático coletado de cistos hidáticos individuais. A internalização do AgB pelas linhagens A549 (célula epitelial de pulmão), NIH/3T3 (fibroblasto), RH (hepatócito) e J774 (macrófago) foi avaliada por imunofluorescência. A viabilidade celular foi analisada pelo ensaio de redução do MTT após incubação com AgB por 24 h. As quatro linhagens celulares foram capazes de internalizar o AgB a 37°C. Para avaliar o envolvimento de endocitose na internalização, células RH foram incubadas com AgB a 4°C, condição que inibe processos de endocitose. Nessa condição, o AgB não foi observado no citoplasma celular, sugerindo que vias de endocitose poderiam estar envolvidas. Células RH e A549 mostraram maior sensibilidade no ensaio de MTT. A taxa de redução de MTT no tratamento com 100 μg/ml de AgB foi de 68% e 57% para RH e A549, respectivamente. Nas mesmas condições, células NIH/3T3 e J774 mostraram respostas similares, o nível de redução do MTT foi 80% e 78%, respectivamente. A sensibilidade observada para as células A549 e RH poderia estar relacionada ao organotropismo por pulmões e fígado reportado na hidatidose cística. A internalização pelas células pode ser uma etapa necessária para a função do AgB como carreador de lipídios ou molécula imunomoduladora e durante esse processo, o AgB poderia causar dano celular nos tecidos do hospedeiro. / Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid cyst) of Echinococcus granulosus. The hydatid cyst develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. E. granulosus antigen B (AgB), one of the major antigenic component of hydatid fluid, is an oligomeric lipoprotein composed by small subunits with approximately 8 kDa. In solution, AgB exists as distinct oligomers and aggegates in equilibrium, however oligomers with approximately 160 kDa are prevalent. The AgB function is not completely clear, but two major roles are suggested: as an immunomodulatory molecule and as a lipid carrier. In this study, the ability of AgB to interact with different cell types and to affect cell viability was investigated. AgB was purified by immunoaffinity chromatography from hydatid fluid collected from individual hydatid cysts. The internalization of AgB by A549 (lung epithelial cell), NIH/3T3 (fibroblast), RH (hepatocyte) and J774 (macrophage) cells was evaluated by immunofluorescence. Cell viability was analyzed by MTT reduction assay after incubation with AgB by 24 h. The four cell lines tested were able to internalize AgB at 37°C. In order to evaluate the role of endocytosis in the internalization, RH cells were incubated with AgB at 4°C, condition that inhibits endocytosis processes. In this condition, AgB was not visualized on cell cytoplasm, suggesting that endocytosis pathways could be envolved. RH and A549 cells showed higher sensitivity on MTT assay. The MTT reduction level on the 100 μg/ml AgB treatment was 68% and 57% for RH and A549, respectively. In the same condition, NIH/3T3 e J774 cells showed similar responses, the MTT reduction level was 80% and 78%, respectively. The observed sensitivity on A549 and RH cells could be related to the organothropism by lungs and liver reported on cystic hydatid disease. The internalization by cells might be a necessary step to the AgB function as a lipid carrier or an immunomodulatory molecule and during this process, AgB could cause cell damage to the host tissues.

A hidatidose cística é uma zoonose causada pelo estágio larval (cisto hidático) do Echinococcus granulosus. O cisto hidático desenvolve-se nas vísceras dos hospedeiros intermediários como uma estrutura unilocular preenchida pelo líquido hidático, que contém os produtos de excreção/secreção do parasito. O antígeno B (AgB) de E. granulosus, um dos principais componentes antigênicos do líquido hidático, é uma lipoproteína oligomérica composta por subunidades de 8 kDa. Em solução o AgB existe como distintos oligômeros e agregados em equilíbrio, contudo, os oligômeros de aproximadamente 160 kDa são predominantes. A função do AgB não é totalmente clara, mas dois papeis principais são sugeridos: de molécula imunomoduladora e de carreador de lipídios. Neste estudo, nós investigamos se o AgB é capaz de interagir com diferentes tipos celulares e afetar sua viabilidade. O AgB foi purificado por cromatografia de imunoafinidade a partir de líquido hidático coletado de cistos hidáticos individuais. A internalização do AgB pelas linhagens A549 (célula epitelial de pulmão), NIH/3T3 (fibroblasto), RH (hepatócito) e J774 (macrófago) foi avaliada por imunofluorescência. A viabilidade celular foi analisada pelo ensaio de redução do MTT após incubação com AgB por 24 h. As quatro linhagens celulares foram capazes de internalizar o AgB a 37°C. Para avaliar o envolvimento de endocitose na internalização, células RH foram incubadas com AgB a 4°C, condição que inibe processos de endocitose. Nessa condição, o AgB não foi observado no citoplasma celular, sugerindo que vias de endocitose poderiam estar envolvidas. Células RH e A549 mostraram maior sensibilidade no ensaio de MTT. A taxa de redução de MTT no tratamento com 100 μg/ml de AgB foi de 68% e 57% para RH e A549, respectivamente. Nas mesmas condições, células NIH/3T3 e J774 mostraram respostas similares, o nível de redução do MTT foi 80% e 78%, respectivamente. A sensibilidade observada para as células A549 e RH poderia estar relacionada ao organotropismo por pulmões e fígado reportado na hidatidose cística. A internalização pelas células pode ser uma etapa necessária para a função do AgB como carreador de lipídios ou molécula imunomoduladora e durante esse processo, o AgB poderia causar dano celular nos tecidos do hospedeiro. / Cystic hydatid disease is a zoonosis caused by the larval stage (hydatid cyst) of Echinococcus granulosus. The hydatid cyst develops in the viscera of intermediate host as a unilocular structure filled by the hydatid fluid, which contains parasitic excretory/secretory products. E. granulosus antigen B (AgB), one of the major antigenic component of hydatid fluid, is an oligomeric lipoprotein composed by small subunits with approximately 8 kDa. In solution, AgB exists as distinct oligomers and aggegates in equilibrium, however oligomers with approximately 160 kDa are prevalent. The AgB function is not completely clear, but two major roles are suggested: as an immunomodulatory molecule and as a lipid carrier. In this study, the ability of AgB to interact with different cell types and to affect cell viability was investigated. AgB was purified by immunoaffinity chromatography from hydatid fluid collected from individual hydatid cysts. The internalization of AgB by A549 (lung epithelial cell), NIH/3T3 (fibroblast), RH (hepatocyte) and J774 (macrophage) cells was evaluated by immunofluorescence. Cell viability was analyzed by MTT reduction assay after incubation with AgB by 24 h. The four cell lines tested were able to internalize AgB at 37°C. In order to evaluate the role of endocytosis in the internalization, RH cells were incubated with AgB at 4°C, condition that inhibits endocytosis processes. In this condition, AgB was not visualized on cell cytoplasm, suggesting that endocytosis pathways could be envolved. RH and A549 cells showed higher sensitivity on MTT assay. The MTT reduction level on the 100 μg/ml AgB treatment was 68% and 57% for RH and A549, respectively. In the same condition, NIH/3T3 e J774 cells showed similar responses, the MTT reduction level was 80% and 78%, respectively. The observed sensitivity on A549 and RH cells could be related to the organothropism by lungs and liver reported on cystic hydatid disease. The internalization by cells might be a necessary step to the AgB function as a lipid carrier or an immunomodulatory molecule and during this process, AgB could cause cell damage to the host tissues.

Résumé : LRP-1 est un récepteur d’endocytose qui fut d’abord associé à des propriétés anti-tumorales via l’internalisation et le catabolisme de protéases matricielles. Cependant, malgré ses capacités à limiter le remodelage de la matrice extracellulaire, LRP-1 peut également coordonner la balance adhérence/dé-adhérence des cellules tumorales afin de favoriser l’invasion. LRP-1 fonctionne ainsi en régulant l’organisation du cytosquelette et le renouvellement des structures d’adhérence grâce à l’activation de la voie MEK/ERK et l’inhibition concomitante de la voie MKK7/JNK. Au cours de ce travail, nous avons cherché à déterminer comment LRP-1 peut réguler le protéome membranaire des cellules tumorales. Nos données révèlent que le taux d’intégrine β1 à la surface de carcinome thyroïdien FTC-133 est augmenté en présence de RAP, un antagoniste de LRP-1. Des immunoprécipitations et des analyses par imagerie confocale montrent que LRP-1 et l’intégrine β1 coexistent au sein des mêmes complexes biomoléculaires. Des tests d’endocytose démontrent que LRP-1 constitue un récepteur d’endocytose de l’intégrine β1 dans les FTC 133 car le nombre d’endosomes contenant l’intégrine β1 est diminué de 30% quand l’endocytose dépendante de LRP-1 est inhibée. Par ailleurs, nos données indiquent que LRP-1 est principalement impliqué dans le recyclage de l’intégrine β1 mais pas dans son ciblage au lysosome. Nous avons ainsi identifié une relation moléculaire privilégiée et originale entre LRP-1 et l’intégrine β1 dans le contexte tumoral. Ces travaux nous ont également incités à initier le développement d’un traceur bimodal original (fluorescence/Raman) permettant de suivre l’endocytose dépendante de LRP 1. / LRP-1 is a large multifunctional endocytic receptor first associated to anti-tumor properties by carrying the uptake and catabolism of extracellular matrix-associated proteinases. However, despite its ability to limit extracellular matrix remodeling, LRP-1 may also coordinate the adhesion/deadhesion balance in malignant cells to support invasion. LRP-1 acts so by regulating the cytoskeleton organization and adhesion structure turnover through the activation of MEK/ERK and concomitant inhibition of MKK7/JNK pathway.During this study, we investigated how LRP-1 is able to regulate the cell-surface proteome in malignant cells. Our data revealed that β1-integrin level is significantly increased at the cell surface of FTC-133 thyroid carcinoma upon treatment with RAP, used as LRP-1 antagonist. Immunoprecipitation experiments and confocal analysis highlight that LRP-1 and β1 integrin coexist at the same biomolecular complexes. Biochemical endocytosis assays demonstrate LRP-1 as a mediator of β1-integrin endocytosis in FTC-133 because the number of endosomes-containing β1-integrin decreases by 30% when LRP-1-mediated endocytosis is inhibited. Moreover, our data indicate that LRP-1 is mainly involved in β1-integrin recycling, but not in lysosome targeting.Overall, we identified an original molecular way between LRP-1 and β1-integrin in the tumor context. These works also allowed to initiate the development of an original bimodal molecular tracker (using both fluorescence and Raman) to study LRP-1-mediated endocytosis.

Protein quality control is crucial to maintaining cellular function. A failure to clear misfolded, aggregation prone proteins can lead to the accumulation of toxic protein aggregates that interfere with cellular pathways and lead to cell death. In addition, the degradation of partially functional proteins can lead to loss of function diseases. Understanding proteins quality control mechanisms is therefore of fundamental importance to understanding these disease pathways. Systems that operate to monitor the structure of soluble protein domains are now relatively well understood. However, in addition to soluble domains, membrane proteins contain regions that span lipid bilayers, and a key question that remains is where and how these transmembrane domains (TMDs) that fail to assemble correctly or are otherwise aberrant are recognised within subcellular compartments. As such, in this study model chimeric proteins containing the luminal and cytoplasmic domain of the single-spanning membrane protein CD8 and exogenous TMDs derived from polytopic membrane proteins were used to investigate the handling of non-native TMDs in the secretory pathway. CD8 chimeras containing non-native TMDs were found to be recognised by endoplasmic reticulum (ER) quality control pathways. Importantly, ER-associated degradation of CD8 chimeras containing exogenous TMDs was reliant upon ubiquitination of cytoplasmic lysine residues prior to retrotranslocation and dislocation from the ER membrane. In contrast, CD8 containing the endogenous TMD but a misfolded luminal domain could be efficiently degraded when cytoplasmic lysines were removed, suggesting that the retrotranslocation mechanisms for these proteins are distinct and defined by the domain which is misfolded. A proportion of the CD8 chimeras containing non-native TMDs were able to exit the ER, and were retrieved to the ER from the Golgi. Golgi-to-ER retrieval was found to be at least partially mediated by Rer1. CD8 chimeras that escaped ER retrieval could also be retained in the Golgi and subsequently degraded in lysosomes, indicating the presence of an as yet undefined TMD-based Golgi quality control checkpoint in mammalian cells. Furthermore, in contrast to WT CD8 which was stable at the plasma membrane, CD8 chimeras containing non-native TMDs that trafficked to the cell surface were rapidly internalised and sorted to lysosomes. This process was largely independent of the cytoplasmic domain of CD8, suggesting signals within the TMD induced internalisation of these CD8 chimeras. The proportion of the CD8 chimeras that trafficked to the plasma membrane, and the stability of the protein at the cell surface, was dependent upon the presence of polar residues within the TMDs, indicating that exposed polar residues in non-native TMDs may alter the handling of proteins at the Golgi and cell surface. Together, these results further our understanding of the mechanisms by which proteins containing aberrant transmembrane domains are handled at multiple subcellular compartments.