BRIEF REPORT |
Correspondence to: Jeffrey L. Brodsky, 274 Crawford Hall, Dept. of Biological Sciences, U. of Pittsburgh, Pittsburgh, PA 15260. E-mail: jbrodsky@pitt.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() |
---|
Almost all secreted proteins pass through the endoplasmic reticulum (ER), an organelle that is equipped to tolerate and/or degrade misfolded proteins. We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP. We propose that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation. (J Histochem Cytochem 51:545548, 2003)
Key Words: Russell bodies, aggresomes, ER, CFTR, BiP, chaperone, yeast, ERAD
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() |
---|
APPROXIMATELY 80% of individuals with cystic fibrosis (CF) have mutations in CFTR that prevent its transport from the ER, and the absence of CFTR at the plasma membrane of epithelial cells results in CF. Most of these mutations prevent CFTR folding, thus converting the protein into a substrate for ER-associated degradation (ERAD), which in turn ensures that only folded proteins progress through the secretory pathway (75% of wild-type CFTR is also subjected to ERAD (
Among their many functions, molecular chaperones catalyze protein degradation. To determine whether ER-associated chaperones are required for wild-type CFTR degradation, we expressed the protein in the yeast S. cerevisiae and reported that the activity of the ER luminal Hsp70 chaperone BiP was dispensable for CFTR degradation, although indirect immunofluorescence (IF) microscopy suggested that CFTR resided in foci that co-localized with BiP (
To maximize epitope accessibility, cryosections of yeast expressing hemaglutinin (HA)-tagged wild-type CFTR were prepared with minimal fixation (
BiP resided in intracellular membranes (Fig 1, large particles) and on occasion CFTR (small particles) co-localized with BiP (e.g., Fig 1A) at what may be ER translocation sites (72% of the CFTR-corresponding particles were clustered in regions lacking BiP. Because CFTR was membrane-associated or in more densely staining membrane-enriched regions, we conclude that CFTR does not reside in "aggresomes" in yeast, which are perinuclear cytoplasmic protein aggregates (
|
|
To further examine BiP and CFTR residency, we fractionated yeast membranes on sucrose density gradients (
|
|
On the basis of these data, we propose that CFTR concentrates at foci in the ER membrane but does not necessarily co-localize with BiP. This is in contrast to BiP bodies that form as a result of an ER-to-Golgi secretion block and that represent BiP-enriched, ER exit sites (
![]() |
Acknowledgments |
---|
Supported by a RDP grant to the University of Pittsburgh from the Cystic Fibrosis Foundation, by grant DK60385-01 from the National Institutes of Health, and by grant MCB-0110331 from the National Science Foundation.
We thank Y. Zhang for supplying yeast used for the initial micrographs, and E. Jones for providing anti-Pep12p antiserum.
Received for publication June 25, 2002; accepted November 27, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() |
---|
Becherer KA, Rieder SE, Emr SD, Jones EW (1996) Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol Biol Cell 7:579-594[Abstract]
Brodsky JL, Hamamoto S, Feldheim D, Schekman R (1993) Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic hsc70. J Cell Biol 120:95-102[Abstract]
Byers TJ, Kunkel LM, Watkins SC (1991) The subcellular distribution of dystrophin in mouse skeletal, cardiac, and smooth muscle. J Cell Biol 115:411-421[Abstract]
Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR et al. (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63:827-834[Medline]
Fewell SW, Travers KJ, Weissman JS, Brodsky JL (2001) The action of molecular chaperones in the early secretory pathway. Annu Rev Genet 35:149-191[Medline]
Hong E, Davidson AR, Kaiser CA (1996) A pathway for targeting soluble misfolded proteins to the yeast vacuole. J Cell Biol 135:623-633[Abstract]
Kopito RR, Sitia R (2000) Aggresomes and Russell bodies. EMBO Rep 1:225-231
Nishikawa S, Hirata A, Nakano A (1994) Inhibition of endoplasmic reticulum (ER)-to-Golgi transport induces relocalization of binding protein (BiP) within the ER to form BiP bodies. Mol Biol Cell 5:1129-1143[Abstract]
Umebayashi K, Hirata A, Fukuda R, Horiuchi H, Ohta A, Takagi M (1997) Accumulation of misfolded protein aggregates leads to the formation of a Russell body-like dilated endoplasmic reticulum in yeast. Yeast 13:1009-1020[Medline]
Wright R (2000) Transmission electron microscopy of yeast. Microsc Res Techn 51:496-510[Medline]
Zhang Y, Nijbroek G, Sullivan ML, McCracken AA, Watkins SC, Michaelis S, Brodsky JL (2001) The Hsp70 molecular chaperone facilitates the ER associated degradation of the cystic fibrosis transmembrane conductance regulator in yeast. Mol Biol Cell 12:1303-1314