Early Secretory Pathway Research Articles

Regulation of Cx36 trafficking through the early secretory pathway by COPII cargo receptors and Grasp55

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing neuron activities or supporting network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif “SAYV” mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system.

Molecular basis of TMED9 oligomerization and entrapment of misfolded protein cargo in the early secretory pathway.

Intracellular accumulation of misfolded proteins causes serious human proteinopathies. The transmembrane emp24 domain 9 (TMED9) cargo receptor promotes a general mechanism of cytotoxicity by entrapping misfolded protein cargos in the early secretory pathway. However, the molecular basis for this TMED9-mediated cargo retention remains elusive. Here, we report cryo-electron microscopy structures of TMED9, which reveal its unexpected self-oligomerization into octamers, dodecamers, and, by extension, even higher-order oligomers. The TMED9 oligomerization is driven by an intrinsic symmetry mismatch between the trimeric coiled coil domain and the tetrameric transmembrane domain. Using frameshifted Mucin 1 as an example of aggregated disease-related protein cargo, we implicate a mode of direct interaction with the TMED9 luminal Golgi-dynamics domain. The structures suggest and we confirm that TMED9 oligomerization favors the recruitment of coat protein I (COPI), but not COPII coatomers, facilitating retrograde transport and explaining the observed cargo entrapment. Our work thus reveals a molecular basis for TMED9-mediated misfolded protein retention in the early secretory pathway.

Zosuquidar Promotes Antitumor Immunity by Inducing Autophagic Degradation of PD-L1.

The intracellular distribution and transportation process are essential for maintaining PD-L1 (programmed death-ligand 1) expression, and intervening in this cellular process may provide promising therapeutic strategies. Here, through a cell-based high content screening, it is found that the ABCB1 (ATP binding cassette subfamily B member 1) modulator zosuquidar dramatically suppresses PD-L1 expression by triggering its autophagic degradation. Mechanistically, ABCB1 interacts with PD-L1 and impairs COP II-mediated PD-L1 transport from ER (endoplasmic reticulum) to Golgi apparatus. The treatment of zosuquidar enhances ABCB1-PD-L1 interaction and leads the ER retention of PD-L1, which is subsequently degraded in the SQSTM1-dependent selective autophagy pathway. In CT26 mouse model and a humanized xenograft mouse model, zosuquidar significantly suppresses tumor growth and accompanies by increased infiltration of cytotoxic T cells. In summary, this study indicates that ABCB1 serves as a negative regulator of PD-L1, and zosuquidar may act as a potential immunotherapy agent by triggering PD-L1 degradation in the early secretory pathway.

Thrombomodulin (p.Cys537Stop) is released from cells by an unusual membrane insertion/leakage mechanism

AbstractExpression of the thrombomodulin (TM) variant c.1611C>A (p.Cys537Stop) leads to the synthesis of a protein with no cytoplasmic tail and a transmembrane domain shortened by 3 amino acids (TM536). However, little is known regarding the release mechanism and properties of TM536. Using umbilical vein endothelial cells and peripheral blood-derived endothelial colony-forming cells from a heterozygous carrier of the TM536 variant as well as overexpression cell models, we demonstrated that TM536 is released from cells by an unusual mechanism. First, TM536 is inserted into the endoplasmic reticulum (ER) membrane, then, because of the low hydrophobicity of its intramembrane domain, it escapes from it and follows the conventional secretory pathway to be released into the extracellular compartment without the involvement of proteolysis. This particular secretion mechanism yields a soluble TM536, which is poorly modified by chondroitin sulfate glycosaminoglycan compared with conventionally secreted soluble forms of TM, and therefore has a suboptimal capacity to mediate thrombin-dependent activation of protein C (PC). We also showed that TM536 cellular trafficking was altered, with retention in the early secretory pathway and increased sensitivity to ER-associated degradation. As expected, activation of ER-associated degradation increased TM536 degradation and reduced its release. The expression of TM536 at the cell surface was low, and its distribution in lipid raft-like membrane microdomains was altered, resulting in low thrombin-dependent PC activation on the cell surface.

CHC22 clathrin recruitment to the early secretory pathway requires two-site interaction with SNX5 and p115

The two clathrin isoforms, CHC17 and CHC22, mediate separate intracellular transport routes. CHC17 performs endocytosis and housekeeping membrane traffic in all cells. CHC22, expressed most highly in skeletal muscle, shuttles the glucose transporter GLUT4 from the ERGIC (endoplasmic-reticulum-to-Golgi intermediate compartment) directly to an intracellular GLUT4 storage compartment (GSC), from where GLUT4 can be mobilized to the plasma membrane by insulin. Here, molecular determinants distinguishing CHC22 from CHC17 trafficking are defined. We show that the C-terminal trimerization domain of CHC22 interacts with SNX5, which also binds the ERGIC tether p115. SNX5, and the functionally redundant SNX6, are required for CHC22 localization independently of their participation in the endosomal ESCPE-1 complex. In tandem, an isoform-specific patch in the CHC22 N-terminal domain separately mediates binding to p115. This dual mode of clathrin recruitment, involving interactions at both N- and C-termini of the heavy chain, is required for CHC22 targeting to ERGIC membranes to mediate the Golgi-bypass route for GLUT4 trafficking. Interference with either interaction inhibits GLUT4 targeting to the GSC, defining a bipartite mechanism regulating a key pathway in human glucose metabolism.

Short transmembrane domains target type II proteins to the Golgi apparatus and type I proteins to the endoplasmic reticulum.

Transmembrane domains (TMDs) contain information targeting membrane proteins to various compartments of the secretory pathway. In previous studies, short or hydrophilic TMDs have been shown to target membrane proteins either to the endoplasmic reticulum (ER) or to the Golgi apparatus. However, the basis for differential sorting to the ER and to the Golgi apparatus remained unclear. To clarify this point, we quantitatively analyzed the intracellular targeting of a collection of proteins exhibiting a single TMD. Our results reveal that membrane topology is a major targeting element in the early secretory pathway: type I proteins with a short TMD are targeted to the ER, and type II proteins to the Golgi apparatus. A combination of three features accounts for the sorting of simple membrane proteins in the secretory pathway: membrane topology, length and hydrophilicity of the TMD, and size of the cytosolic domain. By clarifying the rules governing sorting to the ER and to the Golgi apparatus, our study could revive the search for sorting mechanisms in the early secretory pathway.

The role of the AP-1 adaptor complex in outgoing and incoming membrane traffic.

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.

SNARE chaperone Sly1 directly mediates close-range vesicle tethering.

The essential Golgi protein Sly1 is a member of the Sec1/mammalian Unc-18 (SM) family of SNARE chaperones. Sly1 was originally identified through remarkable gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory but also acts as a positive effector. An amphipathic lipid packing sensor (ALPS)-like helix within the loop directly binds high-curvature membranes. Membrane binding is required for relief of Sly1 autoinhibition and also allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 mutation bypasses requirements for diverse tethering factors but loses this ability if the tethering activity is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to initiate trans-SNARE complex assembly and fusion in the early secretory pathway.

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification.

Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that in Arabidopsis thaliana, GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Unexpectedly, neither single nor double mutants lacking both GRXs isoforms in the cytosol or the ER show phenotypes that differ from wild-type controls. Analysis of electrostatic surface potentials and clustering of GRXs based on their electrostatic interaction with roGFP2 mirrors the phylogenetic classification of class I GRXs, which clearly separates the cytosolic GRXC1 and GRXC2 from the luminal GRXC3 and GRXC4. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC3 and GRXC4 being better catalysts than GRXC1 and GRXC2 for the reduction of bis(2-hydroxyethyl) disulfide. With oxidized roGFP2 as an alternative substrate, GRXC1 and GRXC2 catalyze the reduction faster than GRXC3 and GRXC4, which suggests that catalytic efficiency of GRXs in reductive reactions depends on the respective substrate. Vice versa, GRXC3 and GRXC4 are faster than GRXC1 and GRXC2 in catalyzing the oxidation of pre-reduced roGFP2 in the reverse reaction.

Arabidopsis SEC13B Interacts with Suppressor of Frigida 4 to Repress Flowering.

SECRETORY13 (SEC13) is an essential member of the coat protein complex II (COPII), which was reported to mediate vesicular-specific transport from the endoplasmic reticulum (ER) to the Golgi apparatus and plays a crucial role in early secretory pathways. In Arabidopsis, there are two homologous proteins of SEC13: SEC13A and SEC13B. SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes a C2H2-type zinc finger protein that inhibits flowering by transcriptionally activating the FLOWERING LOCUS C (FLC) through the FRIGIDA (FRI) pathway in Arabidopsis. However, it remains unclear whether SEC13 proteins are involved in Arabidopsis flowering. In this study, we first identified that the sec13b mutant exhibited early flowering under both long-day and short-day conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that both SEC13A and SEC13B were expressed in all the checked tissues, and transient expression assays indicated that SEC13A and SEC13B were localized not only in the ER but also in the nucleus. Then, we identified that SEC13A and SEC13B could interact with SUF4 in vitro and in vivo. Interestingly, both sec13b and suf4 single mutants flowered earlier than the wild type (Col-0), whereas the sec13b suf4 double mutant flowered even earlier than all the others. In addition, the expression of flowering inhibitor FLC was down-regulated, and the expressions of flowering activator FLOWERING LOCUS T (FT), CONSTANS (CO), and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) were up-regulated in sec13b, suf4, and sec13b suf4 mutants, compared with Col-0. Taken together, our results indicated that SEC13B interacted with SUF4, and they may co-regulate the same genes in flowering-regulation pathways. These results also suggested that the COPII component could function in flowering in Arabidopsis.

Metalation and activation of Zn2+ enzymes via early secretory pathway-resident ZNT proteins.

Zinc (Zn2+), an essential trace element, binds to various proteins, including enzymes, transcription factors, channels, and signaling molecules and their receptors, to regulate their activities in a wide range of physiological functions. Zn2+ proteome analyses have indicated that approximately 10% of the proteins encoded by the human genome have potential Zn2+ binding sites. Zn2+ binding to the functional site of a protein (for enzymes, the active site) is termed Zn2+ metalation. In eukaryotic cells, approximately one-third of proteins are targeted to the endoplasmic reticulum; therefore, a considerable number of proteins mature by Zn2+ metalation in the early secretory pathway compartments. Failure to capture Zn2+ in these compartments results in not only the inactivation of enzymes (apo-Zn2+ enzymes), but also their elimination via degradation. This process deserves attention because many Zn2+ enzymes that mature during the secretory process are associated with disease pathogenesis. However, how Zn2+ is mobilized via Zn2+ transporters, particularly ZNTs, and incorporated in enzymes has not been fully elucidated from the cellular perspective and much less from the biophysical perspective. This review focuses on Zn2+ enzymes that are activated by Zn2+ metalation via Zn2+ transporters during the secretory process. Further, we describe the importance of Zn2+ metalation from the physiopathological perspective, helping to reveal the importance of understanding Zn2+ enzymes from a biophysical perspective.

TTC17 is an endoplasmic reticulum resident TPR-containing adaptor protein

Protein folding, quality control, maturation, and trafficking are essential processes for proper cellular homeostasis. Around one third of the human proteome is targeted to the endoplasmic reticulum (ER), the organelle that serves as entrance into the secretory pathway. Successful protein trafficking is paramount for proper cellular function and to that end there are many ER resident proteins that ensure efficient secretion. Here, biochemical and cell biological analysis was used to determined that TTC17 is a large, soluble, ER localized protein that plays an important role in secretory trafficking. Transcriptional analysis identified the predominantly expressed protein isoform of TTC17 in various cell lines. Further, TTC17 localizes to the ER and interacts with a wide variety of chaperones and co-chaperones normally associated with ER protein folding, quality control, and maturation processes. TTC17 was found to be significantly upregulated by ER stress and through the creation and use of TTC17-/- cell lines, quantitative mass spectrometry identified secretory pathway wide trafficking defects in the absence of TTC17. Notably, trafficking of IGF1R, GPNMB, CLU and UGGT1 were significantly altered in H4 neuroglioma cells. This study defines a novel ER trafficking factor and provides insight into the protein-protein assisted trafficking in the early secretory pathway.

DYRK3 enables secretory trafficking by maintaining the liquid-like state of ER exit sites

The dual-specificity kinase DYRK3 controls the formation and dissolution of multiple biomolecular condensates, regulating processes including stress recovery and mitotic progression. Here, we report that DYRK3 functionally interacts with proteins associated with endoplasmic reticulum (ER) exit sites (ERESs) and that inhibition of DYRK3 perturbs the organization of the ERES-Golgi interface and secretory trafficking. DYRK3-mediated regulation of ERES depends on the N-terminal intrinsically disordered region (IDR) of the peripheral membrane protein SEC16A, which co-phase separates with ERES components to form liquid-like condensates on the surface of the ER. By modulating the liquid-like properties of ERES, we show that their physical state is essential for functional cargo trafficking through the early secretory pathway. Our findings support a mechanism whereby phosphorylation by DYRK3 and its reversal by serine-threonine phosphatases regulate the material properties of ERES to create a favorable physicochemical environment for directional membrane traffic in eukaryotic cells.

Anterograde trafficking of Toll-like receptors requires the cargo sorting adaptors TMED-2 and 7.

Toll-Like Receptors (TLRs) play a pivotal role in immunity by recognising conserved structural features of pathogens and initiating the innate immune response. TLR signalling is subject to complex regulation that remains poorly understood. Here we show that two small type I transmembrane receptors, TMED2 and 7, that function as cargo sorting adaptors in the early secretory pathway are required for transport of TLRs from the ER to Golgi. Protein interaction studies reveal that TMED7 interacts with TLR2, TLR4 and TLR5 but not with TLR3 and TLR9. On the other hand, TMED2 interacts with TLR2, TLR4 and TLR3. Dominant negative forms of TMED7 suppress the export of cell surface TLRs from the ER to the Golgi. By contrast TMED2 is required for the ER-export of both plasma membrane and endosomal TLRs. Together, these findings suggest that association of TMED2 and TMED7 with TLRs facilitates anterograde transport from the ER to the Golgi.

The Early Secretory Pathway Is Crucial for Multiple Aspects of the Hepatitis C Virus Life Cycle.

Although most of the early events of the hepatitis C virus (HCV) life cycle are well characterized, our understanding of HCV egress is still unclear. Some reports implicate the conventional endoplasmic reticulum (ER)-Golgi route, while some propose noncanonical secretory routes. Initially, the envelopment of HCV nucleocapsid occurs by budding into the ER lumen. Subsequently, the HCV particle exit from the ER is assumed to be mediated by coat protein complex II (COPII) vesicles. COPII vesicle biogenesis also involves the recruitment of cargo to the site of vesicle biogenesis via interaction with COPII inner coat proteins. We investigated the modulation and the specific role of the individual components of the early secretory pathway in HCV egress. We observed that HCV inhibits cellular protein secretion and triggers the reorganization of the ER exit sites and ER-Golgi intermediate compartments (ERGIC). Gene-specific knockdown of the components of this pathway such as SEC16A, TFG, ERGIC-53, and COPII coat proteins demonstrated the functional significance of these components and the distinct role played by these proteins in various aspects of the HCV life cycle. SEC16A is essential for multiple steps in the HCV life cycle, whereas TFG is specifically involved in HCV egress and ERGIC-53 is crucial for HCV entry. Overall, our study establishes that the components of the early secretory pathway are essential for HCV propagation and emphasize the importance of the ER-Golgi secretory route in this process. Surprisingly, these components are also required for the early stages of the HCV life cycle due to their role in overall intracellular trafficking and homeostasis of the cellular endomembrane system. IMPORTANCE The virus life cycle involves entry into the host, replication of the genome, assembly of infectious progeny, and their subsequent release. Different aspects of the HCV life cycle, including entry, genome replication, and assembly, are well characterized; however, our understanding of the HCV release is still not clear and subject to debate due to varied findings. Here, we attempted to address this controversy and enhance our understanding of HCV egress by evaluating the role of the different components of the early secretory pathway in the HCV life cycle. To our surprise, we found that the components of the early secretory pathway are not only essential for HCV release but also contribute to many other earlier events of the HCV life cycle. This study emphasizes the importance of the early secretory pathway for the establishment of productive HCV infection in hepatocytes.

#3632 CHARACTERIZATION AND REGULATION OF THE PATHOGENIC MUCIN 1 FRAMESHIFT PROTEIN IN PATIENT DERIVED CELLS AFFECTED BY ADTKD-MUC1

Abstract Background and Aims Autosomal Dominant Tubulointerstitial Kidney Diseases (ADTKD) are caused by mutations in one of at least five genes (UMOD, MUC1, HNF1B, REN and SEC61A1) and are characterized by progressive decline in kidney function, reaching end-stage renal disease (ESRD) usually in mid adulthood. ADTKD-MUC1 is one of the most frequent subforms, exclusively caused by a characteristic frameshift mutation, leading to the de novo protein MUC1-fs. MUC1-fs is believed to play the distinct pathogenic role in terms of a toxic proteinopathy, accumulating in the early secretory pathway. The substance BRD4780 is reported to re-route MUC1-fs towards lysosomal degradation (Dvela-Levitt et al., Cell 2019), with interventional trials being in preparation. Therefore, we aimed to gain more insights into MUC1-fs temporal and spacial regulative characteristics, comparing pharmacological intervention vs. RNA interference in patient derived tubular cells. Methods Cell culture (HKC-8 cells and patient derived human urinary Primary Tubular Cells, huPTC); siRNA knockdown; Transient transfection; Immunofluorescence; immunogold electron microscopy (EM); Immunoblotting (IB); Lentiviral SV40/Large-T antigen immortalization of huPTC; Generation of a novel polyclonal MUC1-fs antibody with an independent antigen downstream of the VNTR. Results To analyze MUC1-fs protein in more detail, we generated iTCs (immortalized tubular cells) from huPTC of patients with ADTKD-MUC1. Clonal selection of cells was performed to gain immortalized clones with MUC1-fs expression. MUC1-fs mainly localizes to the secretory pathway. Co-localization was only partially observed with TMED9, which localizes to COP vesicles, being involved in protein trafficking within the early secretory pathway. TMED9 negative components of this pathway also showed MUC1-fs staining, such as the Golgi apparatus and Early Endosomes. Immunogold EM of huPTC reveals MUC1-fs expression within the ER and (secretory) vesicles. Ultrastructural analyses of biopsies by EM from ADTKD-MUC1 patients did not show specific protein accumulation, as previously described in ADTKD-UMOD. RNA interference for MUC1 and pharmacological application of BRD4780 was performed in huPTC and iTCs, with detailed description of MUC1-fs regulation kinetics. Both approaches led to strongly reduced MUC1-fs protein levels. MUC1-fs protein revealed a significantly shorter half life than wildtype mucin 1, possibly due to its different cellular distribution. Conclusion Our data confirm and extend previously published information on intracellular MUC1-fs localization and regulation (Dvela-Levitt et al., Cell 2019). The protein kinetics of MUC1-fs appear quite dynamic in terms of synthesis and decay, which should argue for it being a suitable pharmacological target. Both, BRD4780 and RNA interference potently downregulate MUC1-fs. Interestingly, in primary and immortalized patient derived cells MUC1-fs localization is not confined to the early secretory pathway. Should the effect of BRD4780 be restricted to TMED9 positive COP vesicles, this may implicate reduced effectivenes. Therefore, RNA interference may be more efficient, since it reduces MUC1-fs at an earlier stage and is not dependent on intracellular processing.

Condensation of the β-cell secretory granule luminal cargoes pro/insulin and ICA512 RESP18 homology domain.

ICA512/PTPRN is a receptor tyrosine-like phosphatase implicated in the biogenesis and turnover of the insulin secretory granules (SGs) in pancreatic islet beta cells. Previously we found biophysical evidence that its luminal RESP18 homology domain (RESP18HD) forms a biomolecular condensate and interacts with insulin in vitro at close-to-neutral pH, that is, in conditions resembling those present in the early secretory pathway. Here we provide further evidence for the relevance of these findings by showing that at pH 6.8 RESP18HD interacts also with proinsulin-the physiological insulin precursor found in the early secretory pathway and the major luminal cargo of β-cell nascent SGs. Our light scattering analyses indicate that RESP18HD and proinsulin, but also insulin, populate nanocondensates ranging in size from 15 to 300 nm and 10e2 to 10e6 molecules. Co-condensation of RESP18HD with proinsulin/insulin transforms the initial nanocondensates into microcondensates (size >1 μm). The intrinsic tendency of proinsulin to self-condensate implies that, in the ER, a chaperoning mechanism must arrest its spontaneous intermolecular condensation to allow for proper intramolecular folding. These data further suggest that proinsulin is an early driver of insulin SG biogenesis, in a process in which its co-condensation with RESP18HD participates in their phase separation from other secretory proteins in transit through the same compartments but destined to other routes. Through the cytosolic tail of ICA512, proinsulin co-condensation with RESP18HD may further orchestrate the recruitment of cytosolic factors involved in membrane budding and fission of transport vesicles and nascent SGs.

Rab2 stimulates LC3 lipidation on secretory membranes by noncanonical autophagy

The Golgi complex is a highly dynamic organelle that regulates various cellular activities and yet maintains a distinct structure. Multiple proteins participate in Golgi structure/organization including the small GTPase Rab2. Rab2 is found on the cis/medial Golgi compartments and the endoplasmic reticulum-Golgi intermediate compartment. Interestingly, Rab2 gene amplification occurs in a wide range of human cancers and Golgi morphological alterations are associated with cellular transformation. To learn how Rab2 ‘gain of function’ influences the structure/activity of membrane compartments in the early secretory pathway that may contribute to oncogenesis, NRK cells were transfected with Rab2B cDNA. We found that Rab2B overexpression had a dramatic effect on the morphology of pre- and early Golgi compartments that resulted in a decreased transport rate of VSV-G in the early secretory pathway. We monitored the cells for the autophagic marker protein LC3 based on the findings that depressed membrane trafficking affects homeostasis. Morphological and biochemical studies confirmed that Rab2 ectopic expression stimulated LC3-lipidation on Rab2-containing membranes that was dependent on GAPDH and utilized a non-canonical LC3-conjugation mechanism that is nondegradative. Golgi structural alterations are associated with changes in Golgi-associated signalling pathways. Indeed, Rab2 overexpressing cells had elevated Src activity. We propose that increased Rab2 expression facilitates cis Golgi structural changes that are maintained and tolerated by the cell due to LC3 tagging, and subsequent membrane remodeling triggers Golgi associated signaling pathways that may contribute to oncogenesis.

Zinc homeostasis governed by Golgi-resident ZnT family members regulates ERp44-mediated proteostasis at the ER-Golgi interface

Many secretory enzymes acquire essential zinc ions (Zn2+) in the Golgi complex. ERp44, a chaperone operating in the early secretory pathway, also binds Zn2+ to regulate its client binding and release for the control of protein traffic and homeostasis. Notably, three membrane transporter complexes, ZnT4, ZnT5/ZnT6 and ZnT7, import Zn2+ into the Golgi lumen in exchange with protons. To identify their specific roles, we here perform quantitative Zn2+ imaging using super-resolution microscopy and Zn2+-probes targeted in specific Golgi subregions. Systematic ZnT-knockdowns reveal that ZnT4, ZnT5/ZnT6 and ZnT7 regulate labile Zn2+ concentration at the distal, medial, and proximal Golgi, respectively, consistent with their localization. Time-course imaging of cells undergoing synchronized secretory protein traffic and functional assays demonstrates that ZnT-mediated Zn2+ fluxes tune the localization, trafficking, and client-retrieval activity of ERp44. Altogether, this study provides deep mechanistic insights into how ZnTs control Zn2+ homeostasis and ERp44-mediated proteostasis along the early secretory pathway.

Impact of interorganelle coordination between the conventional early secretory pathway and autophagy in cellular homeostasis and stress response.

The conventional early secretory pathway and autophagy are two essential interconnected cellular processes that are crucial for maintaining cellular homeostasis. The conventional secretory pathway is an anabolic cellular process synthesizing and delivering proteins to distinct locations, including different organelles, the plasma membrane, and the extracellular media. On the other hand, autophagy is a catabolic cellular process that engulfs damaged organelles and aberrant cytosolic constituents into the double autophagosome membrane. After fusion with the lysosome and autolysosome formation, this process triggers digestion and recycling. A growing list of evidence indicates that these anabolic and catabolic processes are mutually regulated. While knowledge about the molecular actors involved in the coordination and functional cooperation between these two processes has increased over time, the mechanisms are still poorly understood. This review article summarized and discussed the most relevant evidence about the key molecular players implicated in the interorganelle crosstalk between the early secretory pathway and autophagy under normal and stressful conditions.