[PubMed] [Google Scholar]Knop M, Hauser N, Wolf DH. for both compartments. Taken together, our results reveal an interdependent relationship between two P-type ATPases to maintain homeostasis of the organelles where they Triciribine phosphate (NSC-280594) reside. INTRODUCTION During biosynthesis, nascent secretory proteins first pass the membranes of the endoplasmic reticulum (ER) through a proteinaceous pore called the translocon (Johnson and van Waes, 1999 ). In the lumen, ER chaperones and folding catalysts assist their folding and assembly. Because these factors are found only in the ER, the folding state of proteins is monitored to assure that only properly folded proteins traffic to their sites of function. This mechanism, termed ER quality control, also functions to select irreversibly damaged proteins for degradation. In this mode, misfolded proteins are exported back to the cytosol, presumably through the translocon, where they are ubiquitinated and degraded by the 26S proteasome (for review, see Brodsky and McCracken, 1999 ; Ellgaard and Helenius, 2001 ). Other than the terminal step, termed ER-associated degradation (ERAD), the mechanisms governing ER quality control remain poorly understood. Two pioneering studies used genetic methodologies to unravel the mechanisms underlying the degradation of proteins in the ER. One used the direct approach of screening for mutants defective in the degradation of two model misfolded soluble proteins: mutant carboxypeptidase Y (CPY*; Wolf and Fink, 1975 ) and mutant proteinase A (PrA*; Finger (was allelic to encodes a Ca2+/Mn2+ ion pump of the P-type ATPase family (Durr (Antebi and Fink, 1992 ; Sorin for ERAD is likely due to its role in maintaining normal Ca2+ levels in the ER (Durr gene, encoding an E3 ubiquitin ligase, is required for degrading misfolded proteins and Hmg2p (Bays (revealed its identity as (Cronin was previously identified to confer salt mediated killer toxin (SMKT) sensitivity, but the mechanism of action is usually unknown. Interestingly, encodes a putative P-type ATPase of a class distinct from (Suzuki and Shimma, 1999 ). Nevertheless, phenotypic analysis of mutants suggested a possible role in Ca2+ homeostasis (Cronin (protein processing in the ER), lead to the constitutive activation of the UPR that, Triciribine phosphate (NSC-280594) in turn, is required for their viability. A variety of functions involved in secretory protein biogenesis and ER quality control were revealed by this approach. One mutant, gene as identical to double mutation nearly abolishes activity, suggesting that each protein can partially compensate for the loss of the other. Taken together, our data support a model of two P-type ATPases, one in the ER and another in the Golgi, working together to maintain homeostasis in the two organelles. MATERIALS AND METHODS Plasmids Used in This Study pSM2 is an expression vector with a hemagglutinin (HA) epitope-tagged version of To construct pSM2, the gene was first subcloned into coding sequences by high-fidelity polymerase chain reaction (PCR) using the primers S1 (5-GCACACTTATTCCCACCTGGTCC-3) S2 (5-CATAAAAGCCATGGCTTTAGAGGCAATCTT-3) followed by digestion with terminator in pRS315 cleaved with was subcloned into pRS425 vector. The construction of pDN431 (HA-epitope tagged CPY*) was described previously (Ng knockout construct. Upstream sequences (500 base pairs) of the Triciribine phosphate (NSC-280594) open reading frame were amplified by PCR using T7 and S3 primers Triciribine phosphate (NSC-280594) (S3: 5-GGGTTACCGATTCCTATGT TTC-3) and pSM1 as template. Downstream sequences (447 base pairs) were amplified using T3 and S4 primers (S4: 5-GGGTAAATCTTTTATGTAAGTAC-3). The fragments were digested with gene Rabbit polyclonal to Vitamin K-dependent protein C from pRS303 (Sikorski and Heiter, 1989 ) was inserted into the null strain, pSM5 was digested with into the locus was confirmed by PCR analysis of genomic DNA. Cloning of the PER9 Gene DNY507 cells were transformed with pDN388 (as the only intact open reading frame. p82-R2Cla was found to complement all mutant phenotypes of and the null strain. Cell Labeling and Immunoprecipitation Typically, 2 mutants. Among these, and were identified as the ERAD-related genes and gene might be novel. Using a colony color sectoring assay, several complementing.
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