Expression of mutant Ins2C96Y results in enhanced tubule formation causing enlargement of pre-Golgi intermediates of CHO cells

Abstract

Misfolded proteins are recognized by the protein quality control and eventually degraded by the ubiquitin-proteasome system. Previously, we demonstrated accumulation of a misfolded non-glycosylated protein, namely proinsulin, in enlarged pre-Golgi intermediates and dilated rough endoplasmic reticulum (ER) domains in pancreatic beta-cells of Akita mice. In order to exclude effects possibly due to coexisting wild type and mutant proinsulin in pancreatic beta-cells, CHO cells expressing singly wild type or mutant C96Y proinsulin 2 were now analyzed by electron microscopic morphometry and immunogold labeling as well as serial section 3D analysis. We found a significant increase in volume density of pre-Golgi intermediates in CHO Ins2(C96Y) cells which was principally due to an increase of its tubular elements, and no significant changes of the ER. The average diameter of the pre-Golgi intermediates of CHO Ins2(C96Y) cells was about twice that of CHO Ins2(wt) cells. The enlarged pre-Golgi intermediates and the ER of CHO Ins2(C96Y) cells were positive for proinsulin, which was not detectable in the significantly enlarged Golgi cisternal stack. Treatment of CHO Ins2(C96Y) cells with proteasome inhibitors resulted in the formation of proinsulin-containing aggresomes. We conclude that misfolded proinsulin causes enlargement of pre-Golgi intermediates which indicates their involvement in protein quality control.

Protein quality control in the early secretory pathway

“…Not only the intrinsic tendency of a protein to polymerize, but also the interactions it establishes in the secretory pathway determine the extent and site of condensation (Mattioli et al , 2006). Accumulation in post‐ER compartments has been described for mutant pro‐insulin in the Akita diabetes model (Zuber et al , 2004; Fan et al , 2007) and vasopressin V2 receptor in nephrogenic diabetes insipidus (Oueslati et al , 2007), in line with the existence of sequential checkpoints. It remains to be seen whether these unusual localizations reflect accumulation in a specialized early secretory pathway subregion, the so‐called ‘quality control compartment’ originally described for ASGPR H2a mutants (Kamhi‐Nesher et al , 2001; Kondratyev et al , 2007).…”

Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted

“…In these studies, we have followed the lead of others who have shown that WT and mutant forms of human proinsulin can be studied using in vitro cell culture systems, including cell lines that do not normally synthesize insulin such as HEK 293 and AtT20 cells as well as those that do, such as INS-1 or other insulinoma cell lines [5,15,21–28]. There are advantages to studying heterologous expression of human proinsulin in multiple types of cells as parallel studies allow comparison of secretion via constitutive (HEK 293 cells) and regulated pathways (INS-1 cells).…”

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“…The authors described in detail mechanisms of the ER-associated degradation (ERAD). Additionally, they detailed degradation mechanisms residing within and beyond the ER, in particular glucosidase II (GlsII), UDPglucose:glycoprotein glucosyltransferase (GT), and calretinin, which were, by immunoelectron microscopy, localized in both the rough and the smooth ER, and in pre-Golgi intermediates which had previously been shown to be involved in protein quality control (Fan et al 2007). Further topics of the review were novel ER exit pathways for aberrant proteins as deWned by the ERAD factor EDEM1; the Wnding that endomannosidase localized in the Golgiapparatus further provides sugar trimming capacity for the maturation of glycoproteins in this compartment; and numerous observations of organelle changes caused by protein folding diseases.…”