7 research outputs found
Inadequate BiP availability defines endoplasmic reticulum stress.
How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible bulk expression of secretory immunoglobulin M heavy chain (μs) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µs is restored lead to µs-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (CH1) from µs. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem
Roles of N-glycans in the polymerization-dependent aggregation of mutant Ig-\u3bc chains in the early secretory pathway
The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-\u3bc chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant \u3bc chains lacking the C\u3bc1 domain (\u3bc\u394). Our data reveal that \u3bc\u394 lacking 563 glycans (\u3bc\u3945) form larger intracellular aggregates than \u3bc\u394 and are not secreted. Like \u3bc\u394, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, \u3bc\u394 lacking 402 glycans (\u3bc\u3944) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (\u3bc\u3944-5). These results suggest that the two C-terminal Ig-\u3bc glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers
Role of Dynactin in Endocytic Traffic: Effects of Dynamitin Overexpression and Colocalization with CLIP-170
The flow of material from peripheral, early endosomes to late endosomes requires microtubules and is thought to be facilitated by the minus end-directed motor cytoplasmic dynein and its activator dynactin. The microtubule-binding protein CLIP-170 may also play a role by providing an early link to endosomes. Here, we show that perturbation of dynactin function in vivo affects endosome dynamics and trafficking. Endosome movement, which is normally bidirectional, is completely inhibited. Receptor-mediated uptake and recycling occur normally, but cells are less susceptible to infection by enveloped viruses that require delivery to late endosomes, and they show reduced accumulation of lysosomally targeted probes. Dynactin colocalizes at microtubule plus ends with CLIP-170 in a way that depends on CLIP-170’s putative cargo-binding domain. Overexpression studies using p150(Glued), the microtubule-binding subunit of dynactin, and mutant and wild-type forms of CLIP-170 indicate that CLIP-170 recruits dynactin to microtubule ends. These data suggest a new model for the formation of motile complexes of endosomes and microtubules early in the endocytic pathway
The intracellular quality control system down-regulates the secretion of amyloidogenic apolipoprotein A-I variants: A possible impact on the natural history of the disease
Hereditary systemic amyloidosis caused by apolipoprotein A-I variants is a
dominantly inherited disease characterised by fibrillar deposits mainly localized
in the kidneys, liver, testis and heart. We have previously shown that the
apolipoprotein A-I variant circulates in plasma at lower levels than the
wild-type form (Mangione et al., 2001; Obici et al., 2004) thus raising the
possibility that the amyloid deposits could sequester the circulating
amyloidogenic chain or that the intracellular quality control can catch and
capture the misfolded amyloidogenic chain before the secretion. In this study we
have measured plasma levels of the wild-type and the variant Leu75Pro
apolipoprotein A-I in two young heterozygous carriers in which tissue amyloid
deposition was still absent. In both cases, the mutant was present at
significantly lower levels than the wild-type form, thus indicating that the low
plasma concentration of the apolipoprotein A-I variant is not a consequence of
the protein entrapment in the amyloid deposits. In order to explore the cell
secretion of amyloidogenic apolipoprotein A-I variants, we have studied COS-7
cells expressing either wild-type apolipoprotein A-I or two amyloidogenic
mutants: Leu75Pro and Leu174Ser. Quantification of intracellular and
extracellular apolipoprotein A-I alongside the intra-cytoplasmatic localization
indicates that, unlike the wild-type protein, both variants are retained within
the cells and mainly accumulate in the endoplasmic reticulum. The low plasma
concentration of amyloidogenic apolipoprotein A-I may therefore be ascribed to
the activity of the intracellular quality control that represents a first line of
defence against the secretion of pathogenic variants
Sequential steps and checkpoints in the early exocytic compartment during secretory IgM biogenesis
The biogenesis of secretory IgM occurs stepwise under stringent quality control, formation of ÎĽ2L2 preceding polymerization. How is efficiency of IgM secretion coupled to fidelity? We show here that ERp44, a soluble protein involved in thiol-mediated retention, interacts with ERGIC-53. Binding to this hexameric lectin contributes to ERp44 localization in the ER-golgi intermediate compartment. ERp44 and ERGIC-53 increase during B-lymphocyte differentiation, concomitantly with the onset of IgM polymerization. Both preferentially bind ÎĽ2L2 and higher order intermediates. Their overexpression or silencing in non-lymphoid cells promotes or decreases secretion of IgM polymers, respectively. In IgM-secreting B-lymphoma cells, ÎĽ chains interact first with BiP and later with ERp44 and ERGIC-53. Our findings suggest that ERGIC-53 provides a platform that receives ÎĽ2L2 subunits from the BiP-dependent checkpoint, assisting polymerization. In this process, ERp44 couples thiol-dependent assembly and quality control