Endoplasmic reticulum-associated degradation of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) protein is known to involve the ubiquitinproteasome system. In addition, an ATP-independent proteolytic system has been suggested to operate in parallel with this pathway and become up-regulated when proteasomes are inhibited (Jensen, T. J., Loo, M. A., Pind, S., Williams, D. B., Goldberg, A. L., and Riordan, J. R. (1995) Cell 83, 129 -135). In this study, we use two independent techniques, pulse-chase labeling and a noninvasive fluorescence cell-based assay, to investigate the proteolytic pathways underlying the degradation of misfolded CFTR. Here we report that only inhibitors of the proteasome have a significant effect on preventing the degradation of CFTR, whereas cell-permeable inhibitors of lysosomal degradation, autophagy, and several classes of protease had no measurable effect on CFTR degradation, when used either alone or in combination with the specific proteasome inhibitor carbobenzoxy-Lleucyl-leucyl-L-leucinal (MG132). Our results suggest that ubiquitin-proteasome-mediated degradation is the dominant pathway for disposal of misfolded CFTR in mammalian cells and provide new mechanistic insight into endoplasmic reticulum-associated degradation.

Cells control organelle size with great precision and accuracy to maintain optimal physiology, but the mechanisms by which they do so are largely unknown. Cilia and flagella are simple organelles in which a single measurement, length, can represent size. Maintenance of flagellar length requires an active transport process known as intraflagellar transport, and previous measurements suggest that a length-dependent feedback regulates intraflagellar transport. But the question remains: how is a length-dependent signal produced to regulate intraflagellar transport appropriately? Several conceptual models have been suggested, but testing these models quantitatively requires that they be cast in mathematical form. Here, we derive a set of mathematical models that represent the main broad classes of hypothetical size-control mechanisms currently under consideration. We use these models to predict the relation between length and intraflagellar transport, and then compare the predicted relations for each model with experimental data. We find that three models-an initial bolus formation model, an ion current model, and a diffusion-based model-show particularly good agreement with available experimental data. The initial bolus and ion current models give mathematically equivalent predictions for length control, but fluorescence recovery after photobleaching experiments rule out the initial bolus model, suggesting that either the ion current model or a diffusion-based model is more likely correct. The general biophysical principles of the ion current and diffusion-based models presented here to measure cilia and flagellar length can be generalized to measure any membrane-bound organelle volume, such as the nucleus and endoplasmic reticulum.

A mutation in a microtubule-severing enzyme, katanin, causes flagella to become short due to a reduced cytoplasmic precursor pool. These results suggest that competition between flagella and cytoplasmic microtubules for a limited tubulin pool is facilitated by katanin, which is confirmed by stochastic models.

No abstract