ABCMdb

PMID: 10225950

Wigley WC, Fabunmi RP, Lee MG, Marino CR, Muallem S, DeMartino GN, Thomas PJ
Dynamic association of proteasomal machinery with the centrosome.
J Cell Biol. 1999 May 3;145(3):481-90., 1999-05-03 [PubMed]

Sentences

No. Mutations Sentence Comment

34 ABCC7 p.Pro205Ser Several other CF-causing folding mutations (Gregory et al., 1991; Sheppard et al., 1996) including P205S which is located in the third membrane spanning helix (Wigley et al., 1998) of TMD1, also result in inefficient processing and maturation and increased degradation. Login to comment 35 ABCC7 p.Pro205Ser Significantly, both the ⌬F508 and P205S folding mutants are functional when they assume a native conformation, thus raising the possibility that overcoming the maturation deficiency by correcting the underlying folding defect or by circumventing a proteolytic recognition step may be of therapeutic benefit. Login to comment 61 ABCC7 p.Pro205Ser Preparation of P205S Mutant Expression Construct Oligonucleotide-directed mutagenesis as described (Andrews and Lesley, 1998) was used to generate the mutant CFTR from the parent expression vector pCMVNot6.2. Login to comment 63 ABCC7 p.Pro205Ser The sequence of the mutagenic primer used to create P205S was 5Ј-CGTGTGGATCGCT- TCTTTGCAAGTGGC-3Ј. Login to comment 103 ABCC7 p.Pro205Ser Analysis of Soluble and Insoluble Cellular Fractions 2.5 ϫ 105 HeLa cells per 3-cm dish were either mock-transfected or transfected with P205S mutant CFTR expression plasmid. Login to comment 152 ABCC7 p.Pro205Ser To further test this hypothesis, we expressed in HEK 293 cells wild-type CFTR and two variants known to misfold, namely ⌬F508 and P205S, and examined their effect on the centrosome and the proteasome machinery. Login to comment 156 ABCC7 p.Pro205Ser In sharp contrast, ⌬F508 and P205S mutant CFTR are detected predominantly in the ER of transfected cells as illustrated by the ER pattern of CFTR staining (Fig. 4, C and E) and the complete colocalization with BiP staining (Fig. 4, D and F). Login to comment 159 ABCC7 p.Pro205Ser In striking contrast, expression of P205S (Fig. 5 B) or ⌬F508 (data not shown) expands the centrosome in a manner similar to that observed when cells are treated with lactacystin alone (Fig. 3). Login to comment 169 ABCC7 p.Pro205Ser Recruitment of Proteasomal Components to a Centrosome-Associated Inclusion In cells overexpressing mutant CFTR (P205S) and treated with lactacystin, we observed the formation of large, perinuclear aggregates of misfolded CFTR which appear to arise from the centrosome as indicated by colocalization with ␥-tubulin (Fig. 6). Login to comment 179 ABCC7 p.Pro205Ser However, in cells expressing P205S mutant CFTR and treated with lactacystin, ␥-tubulin was observed distributed between the soluble and insoluble fractions. Login to comment 186 ABCC7 p.Pro205Ser When the cellular level of misfolded protein is high, either due to the overexpression of a misfolded mutant protein (such as ⌬F508 or P205S CFTR) or the inhibition of the proteasome, the cell responds by expanding the diame- Figure 3. Login to comment 204 ABCC7 p.Pro205Ser Transiently transfected HEK 293 cells expressing either wild-type or mutant P205S CFTR (as indicated) were stained with antibodies against CFTR (red) and either PA28 (A) or 20S proteasome (B) (each in green). Login to comment 226 ABCC7 p.Pro205Ser The expansion due to inhibition of the proteasome with lactacystin alone and lactacystin with low expression of wild-type, ⌬F508, and P205S CFTR was determined from at least eight measurements. Login to comment