Expression of mutant Ins2C96Y results in enhanced tubule formation causing enlargement of pre-Golgi intermediates of CHO cells.
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.
Fan
Jing-Yu
JY
Division of Cell and Molecular Pathology, Department of Pathology, University of Zürich, CH-8091, Zürich, Switzerland, christian.zuber@usz.ch.
Roth
Jürgen
J
Zuber
Christian
C
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure*; D007150:Immunohistochemistry; D051379:Mice; D020125:Mutation, Missense; D011384:Proinsulin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors
D061988:Proteasome Inhibitors; D011384:Proinsulin