the question in the attachment
1-. Protein misfolding can be an aspect of several different human disorders, including cystic fibrosis, Alzheimer's disease, and atherosclerosis. Many times, the misfolded protein is a membrane protein. In fact, a type of diabetes insipidus results from a mutation in the G-protein-coupled vasopressin 2 receptor that prevents the protein from making it to the cell membrane! Describe how this type of receptor would normally get targeted to the membrane (from the beginning of translation) and then propose one mechanism by which the mutation could cause a lack of proper targeting. In the nucleus the RNA is made its process and transported into cytoplasm to mRNA and ribosome, which is the main machinery for translated protein (translation process), bound to mRNA and signal recognition peptide bound to synthesis protein. If the protein has signal sequence, it recognizes by chaperone in the surface of ER that responsible for protein folding inside a vesicle. It will make a chemical modification for the protein by glycosylation, phosphorylation, acetylation etc. Then it will become a mature protein. There are specific receptor in ER and the protein gets made. so that protein visecle start to move cross the ER membrane through ER translocon which is a channel where is G protein coupled receptor pass the membrane for 7 times because of the start and stop signals will leave the channel in and out. (Some of the proteins going to the lumen of the ER that’s different than if the protein stuck in ER. That’s, also, different if the protein has single transmembrane domain, so that has multiple transmembrane domain). When the protein leaves the ER, it goes to the final processing the receptor will bind to COP protein through Golgi complex, where the most of sugar and lipid are added to the proteins. The ER is continuous network membrane, so it can stay or go to the nuclear in and out. Each Golgi layer separate from the other, but the protein moves in the process from inside to outside of Golgi by using little vesicles. Finally, it formed vesicles for the protein after the modification then will fusion to cell membrane. Cystic Fibrosis (CFTR) caused by mutation in Cystic Fibrosis transmembrane conductant regulator. CFTR could happen with many different mutations not only this. It called (deletion of phenylalanine 508 (ΔF508). CFTR gene in chromosome7 its 250,000-base pair long. Most of DNA gene is non-codon introns with 24 exons on pre- mRNA that can splice it into mature mRNA by taking the exons and join them together that it goes it to ribosome to make cystic fibrosis transmembrane regulator polypeptide then translation to get CFTR protein transmembrane domain 1 (TMD1) bind to nucleotide binding domain 1 (NBD1) by the loop. NBD1 bind to regulatory domain and TMD2 bind to NBD2. Finally ending with PDZ domain that will bind to cytoskeleton. Deletion mutation happened in the exon that is deleted 508 phenylalanine in the NBD1. It occurs in the RER and this deletion cause unfolded CFTR protein. So, it will stop from traffic from ER.ER quality control mechanism kicks in and they start protein stop binding to CFTR protein and then retain it in ER and it doesn’t go to Golgi. Eventually, target it, destroyed it and eat it by lysosome. Protein comes in and amino acid comes out then it goes into proteasome and break it up into pieces. finally, it doesn’t end up with that much of CFTR at all in cell membrane. 2-From figure 3 in the paper (1) Diagram the results that you would expect to see in Lanes T, 1, 2, 3, and 4, if the amino acid signal DID in the protein Gap1p was mutated to random amino acids and tell why; and (2) Give two possible (different) results that might occur if the amino acid signal LxxLE in Bet1p was mutated to the amino acids DID (which are the signal in Gap1p). Diagram the results expected in Lanes T, 1, 2, 3, and 4 and explain why you predicted this result for each case?. doi:10.1016/S0092-8674(03)00609-3 Cell, Vol. 114, 497–509, August 22, 2003, Copyright 2003 by Cell Press Multiple Cargo Binding Sites on the COPII Subunit Sec24p Ensure Capture of Diverse Membrane Proteins into Transport Vesicles first vesicular step in forward transport of secretory pro- teins (Barlowe et al., 1994). The process of cargo selec- tion by the COPII coat is thought to be facilitated by the Sec24p subunit. Sec23/24p binds to cargo molecules in vitro (Peng et al., 1999; Springer and Schekman, 1998; Elizabeth A. Miller,1 Traude H. Beilharz,1,2 Per N. Malkus,1,2 Marcus C.S. Lee,1 Susan Hamamoto,1,2 Lelio Orci,3 and Randy Schekman1,2,* 1Department of Molecular and Cell Biology and Votsmeier and Gallwitz, 2001), and cargo-containing2 Howard Hughes Medical Institute “prebudding” complexes can be isolated after incuba-University of California, Berkeley tion of ER membranes with Sar1p and Sec23/24p (AridorBerkeley, California 94720 et al., 1998; Kuehn et al., 1998). Furthermore, a homolog3 Department of Morphology of Sec24p, Lst1p, is specifically required for efficientUniversity of Geneva Medical School packaging of the plasma membrane protein Pma1p1121 Geneva 4 (Roberg et al., 1999; Shimoni et al., 2000). Moreover,Switzerland vesicles generated with Sec23/Lst1p in the absence of Sec24p contain a unique subset of cargo molecules, suggesting distinct cargo specificities for Sec24p andSummary Lst1p (Miller et al., 2002). The sorting signals that govern uptake into COPII vesi-We have characterized the mechanisms of cargo se- cles are relatively poorly defined. In contrast to thelection into ER-derived vesicles by the COPII subunit well-characterized signals that mediate endocytosisSec24p. We identified a site on Sec24p that recognizes and retrograde transport between the Golgi and ER,the v-SNARE Bet1p and show that packaging of a num- there is no apparent universal ER export signal that isber of cargo molecules is disrupted when mutations are present on all secretory proteins. Recent work charac-introduced at this site. Surprisingly, cargo proteins terizing the cytoplasmic domains of a variety of cargoaffected by these mutations did not share a single molecules from diverse systems has revealed twocommon sorting signal, nor were proteins sharing a classes of ER export signals. A dihydrophobic motif,putative class of signal affected to the same degree. often consisting of two C-terminal hydrophobic resi-We show that the same site is conserved as a cargo- dues, facilitates export of the p24 family of proteinsinteraction domain on the Sec24p homolog Lst1p, (Nakamura et al., 1998) as well as the ERGIC53/Emp47pwhich only packages a subset of the cargoes recog- proteins (Kappeler et al., 1997; Sato and Nakano, 2002).nized by Sec24p. Finally, we identified an additional A second class of signal, a diacidic motif, directs ERmutation that defines another cargo binding domain export of the viral, mammalian, and yeast proteins,on Sec24p, which specifically interacts with the SNARE VSV-G, Kir1.1/Kir1.2, and Sys1p, respectively (Nishi-Sec22p. Together, our data support a model whereby mura and Balch, 1997; Stockklausner et al., 2001; Vots-Sec24p proteins contain multiple independent cargo meier and Gallwitz, 2001). However, in the case ofbinding domains that allow for recognition of a diverse VSV-G, additional residues also contribute to efficientset of sorting signals. packaging (Nishimura et al., 1999; Sevier et al., 2000). Many of these ER export signals have been shown toIntroduction bind to the COPII subunit Sec23/24p. Importantly, muta- tion of the sorting motif abrogates both Sec23/24p bind-Transport through the secretory pathway is mediated ing and ER export (Aridor et al., 2001; Belden and Bar-by vectorial transfer of proteins between compartments lowe, 2001a; Nufer et al., 2002).within membrane bound vesicles. Generation of vesicles Here, we describe the characterization of the molecu- is achieved by compartment-specific cytoplasmic coat lar mechanisms of cargo recruitment by the Sec24p proteins that function both in membrane deformation subunit of the COPII coat. Using an alanine-scanning and compartmental organization such that cargo mole- mutagenesis approach, we have identified and charac- cules are included in nascent vesicles, while residents terized mutations in Sec24p that specifically disrupt are excluded. This process of cargo selection is thought recruitment of a subset of cargo molecules. Our data to be driven by direct interaction of coat proteins with suggest that Sec24p has multiple independent sites of specific sorting signals that are responsible for directing cargo recognition. Together with a detailed structural uptake of cargo molecules into a budding vesicle (Aridor and biochemical analysis of coat-SNARE interactions and Traub, 2002; Schekman and Orci, 1996). Different (Mossessova et al., 2003), these functional studies dem- cytoplasmic coat proteins mediate transport between onstrate that cargo recruitment into nascent vesicles is the various compartments of the secretory pathway, driven by direct interaction between a sorting signal each recognizing distinct sorting signals borne by cargo and a coat subunit and provide an important basis for molecules. identifying distinct sorting signals and novel cargo inter- The COPII coat, composed of five proteins that com- action domains. prise three subunits (Sar1p, Sec23/24p, and Sec13/31p), generates vesicles from the endoplasmic reticulum (ER) Results that are destined to fuse with the Golgi apparatus, the The Sec23/24p complex of the COPII coat binds avidly to the cytoplasmic domains of a number of cargo mole-*Correspondence:
[email protected] Cell 498 the wild-type Sys1 peptide (Figure 1A). However, a pep- tide that contained mutations in the residues that make critical contact with Sec23/24p (D198AxE200A) failed to compete with GST-Bet1p for interaction with Sec23/ 24p (Figures 1A and 1C). Having demonstrated a direct competition between Bet1p and Sys1p for interaction with Sec23/24p, we investigated whether an additional cargo molecule with a distinct sorting motif could com- pete for the same site. The C-terminal cytoplasmic do- main of Prm8p contains a diphenylalanine motif that is required for interaction with Sec23/24p (S. Springer and R.S., unpublished data). A peptide corresponding to this region binds avidly to Sec23/24p (S. Springer and R.S., unpublished data) and was tested for its ability to com- pete with GST-Bet1p. Unlike the Sys1p signal, neither the wild-type Prm8p signal, nor a mutant form that lacked the diphenylalanine motif was able to compete with GST-Bet1p for Sec23/24p binding (Figures 1B and 1C). These data suggest that Sys1p and Bet1p bind to the same site on Sec24p, which is likely to have another independent binding site that may interact with addi- tional cargo molecules. Cargo Binding Mutations in Sec24p We undertook a genetic approach to identify functionally important residues on Sec24p that might be involved in cargo selection. The recent structure of the Sec23/ Sec24/Sar1p complex (Bi et al., 2002) allowed the use of targeted alanine-scanning mutagenesis to alter charged residues around the equatorial (i.e., neither membrane proximal nor membrane distal) surface of Sec24p. WeFigure 1. Bet1p and Sys1p Compete for the Same Site on Sec24p screened for mutants that were unable to complement(A) The cytoplasmic domain of Bet1p fused