QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 23 April 2003
doi: 10.1242/jcs.00439

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Palmer, E. A. Articles by McCracken, A. A. Search for Related Content PubMed PubMed Citation Articles by Palmer, E. A. Articles by McCracken, A. A. Social Bookmarking                         What's this?

Differential requirements of novel A1PiZ degradation deficient (ADD) genes in ER-associated protein degradation

¹ Biology Department, University of Nevada, Reno, NV 89557, USA
² Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

View larger version (77K): [in a new window]   Fig. 1. New add mutants identified by colony-blot immunoassay screening. Representative immunoassay performed on each gene deletion strain as described in Materials and Methods. Rows (a-l) show, in duplicate from left to right, colonies of deletion mutants #01-06 (a), 07-12 (b), 13-18 (c), 19-24 (d), 25-30 (e), 31-36 (f), 37-42 (g), 43-48 (h), 49-54 (i), 55-60 (j), 62-67 (k), 68-73 (l). Row (s) shows duplicates of the M, Z and O parent strain standard controls from left to right, expressing wild-type A1PiM or mutant A1PiZ, or the pYES2.0 vector with no gene insertion. Those deletion mutants exhibiting a darker colony spot than that of the BY4742 WT parent expressing A1PiZ were selected as putative ERAD mutants and re-assayed to eliminate false-positive results. Only deletion mutants 06, 37, 39, 66, 67, 68 and 72 displayed a consistent signal darker than the BY4742 WT parent (Z) in subsequent colony-blot immunoassays. For example, deletion mutants 59 and 60 were selected from this assay but were eliminated by at least two negative results in future assays.

View larger version (33K): [in a new window]   Fig. 2. The A1PiZ degradation rate is slowed in the add mutants. Pulse-chase radiolabeling experiments were performed with add mutants and the parent wild type, BY4742, expressing A1PiZ. (A) A1PiZ was immunoprecipitated from the cell extracts at 0, 20, 40 and 60 minutes and resolved on a 10% SDS-PAGE (Methods and Materials). (B) The relative amounts of A1PiZ were determined using the Bio-Rad Phosphor Analyses program with the amount of A1PiZ at the zero time point set at 100%. Results shown are the average of five independent experiments, ±s.d.

View larger version (39K): [in a new window]   Fig. 3. Complementation of the add phenotype. Representative immunoassays performed as described in Materials and Methods. Deletion mutants (add06, add37, add39, add66, add67 and add68) expressing A1PiZ were transformed with expression vectors carrying the corresponding ADD gene (right) or with the appropriate vector without gene insert (left) and duplicate colonies were assayed. Standard controls were BY4742 WT parent stain expressing A1Pi (M), mutant A1PiZ (Z) or the vector with no gene insertion (0).

View larger version (35K): [in a new window]   Fig. 4. CFTR degradation is compromised in the add mutants. CFTR-expressing cells were grown to mid-log phase, cycloheximide was added, and the cells were harvested at the indicated time points. (A) Cell extracts were prepared at 0, 20, 40 and 60 minutes and subjected to SDS-PAGE followed by quantitative immunoblot analysis. (B) The relative amounts of CFTR were determined using the Bio-Rad Phosphor Analyses program with the amount of CFTR at the zero time point set at 100%. Results shown are the average of three independent experiments, ±s.d.

View larger version (18K): [in a new window]   Fig. 5. Microsomes and cytosol prepared from add37, add39, add66 and add67, but neither add06 nor add68 are proficient for pF degradation in vitro. (A) Microsomes containing pF were prepared from each ADD deletion strain and incubated in the (a) absence or (b) presence of cytosol made from the same strain; the amount of pF was determined at 0 and 40 minutes after addition of cytosol or buffer. (B,C) Microsomes and cytosol were prepared from both WT and deletion strains (B, add06 and C, add68) and analyzed for ERAD in vitro in the indicated combinations of microsomes/cytosol: WT/WT; • WT/buffer; add/buffer; add/add; WT/add; add/WT. The amount of pF was determined using the Bio-Rad Phosphor Analyses program. Data represents the mean of triplicate experiments, ±s.d.

View larger version (10K): [in a new window]   Fig. 6. UPR induction in add mutants. Respective isogenic wild type and the kar2-1 and add mutant strains were transformed with the UPR reporter plasmid, and relative activity (compared to the wild type) was assayed as described in the Materials and Methods. Data represent the means of two experiments, each performed with two unique transformants of each strain.

View larger version (49K): [in a new window]   Fig. 7. Cells deleted for ADD68 are hypersensitive to cadmium. Exponentially growing liquid cultures of each ADD deletion strain (66, 67, 68, 06, 37, 39, 43 and 72) and the isogenic parent (WT) were spotted in ten-fold dilutions of 0.001 OD/µl on complete medium (CD) or medium containing cadmium (+15 µM CdCl2) and were incubated at 30oC for 36 hours. Plates were imaged using a Mustek 600-11-CD scanner.

View larger version (77K): [in a new window]   Fig. 8. kar2-1 add37 double mutants display a synthetic growth defect. Exponentially growing cultures of dissected tetrads were plated at tenfold dilutions on complete media and incubated at 37oC for two days. Progeny from crosses add37 X kar2-1 (A) and add67 X kar2-1 (B) were ordered in rows from top to bottom: WT strain, ADD/KAR2; ADD deletion mutant strain, add/KAR2; temperature-sensitive kar2-1 strain, ADD/kar2-1; and the double mutant, add/kar2-1. The results for crosses add06 X kar2-1 and add39 X kar2-1 were similar to that seen for add67 X kar2-1.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter