TANG BOR LUENINSTITUTE OF MOLECULAR & CELL BIOLOGY2020-08-072020-08-071995TANG BOR LUEN (1995). PROTEIN TARGETING AND RETENTION IN THE ENDOPLASMIC RETICULUM AND THE GOLGI APPARATUS. ScholarBank@NUS Repository.https://scholarbank.nus.edu.sg/handle/10635/172051Secretory and membrane proteins en route to the cell surface are first targeted and translocated into the ER followed by their subsequent transport to the Golgi apparatus. Anterograde transport occurs presumably by default and resident proteins of each membrane bound compartment must be either actively retained or passively retarded. The signals and mechanisms of protein targeting and retention in the ER and the Golgi apparatus were investigated. The tetrapeptide sequence KDEL, known previously as the ER retention signal for soluble ER resident proteins, was found also to retain a type II cell surface membrane protein, dipeptidyl peptidase IV (D4) in the ER when appended to it. To further characterize the machinery of ER retention, cDNA of the bovine KDEL receptor was cloned and sequenced. Antibody raised against a C-terminal peptide stretch demonstrated that the KDEL receptor is present ubiquitously in mammalian cells. Immunofluorescence localization revealed that the receptor is found in the perinuclear Golgi region, with spotty stainings distributed throughout the cell. Interestingly, the protein can be shirted from the perinuclear region to the spotty region or vice versa during brefeldin A (BFA) and low temperature treatment or upon recovery from those treatments. During recovery, tubular structures stained by the antibody was observed. The subcellular dynamics of the receptor is consistant with its putative function in the ER retrieval machinery. The anti-KDEL receptor antibody binding was greatly diminished in digitonin permeabilized cells preincubated with eukaryotic cell cytosol and fixed prior· to antibody incubation. Inhibition is prevented by coincubation with the antibody and is dose, temperature and energy dependent and sensitive to N-ethylmaleimide. The findings are suggestive of the existence of cytosolic factor (s) which interacts with the cytoplasmic C-terminus of the KDEL receptor, which are likely to be components of the KDEL protein retrieval machinery. The enzyme N-acetylglucosaminyltransferase I (NT, EC 2.4. l. 101) is a resident type II transmembrane protein of the Golgi apparatus. To delineate the portion of its primary sequence that is responsible for the Golgi retention of this protein, chimeras containing different N-terminal portions of NT joined to a reporter sequence, the ectodomain of a type II surface membrane protein, were constructed. The transmembrane domain of NT was found to be sufficient to confer Golgi retention of the fusion proteins. Chimeras of the cell surface protein dipeptidyl peptidase IV containing the transmembrane domain of ?-galactoside ?2,6-sialyltransferase (ST) and N-acetylglucosaminyltransferase 1 (NT) are retained in the Golgi apparatus in MDCK and COS cells, as assessed by indirect immunofluorescence microscopy. Transfection of these chimeric constructs into CHO cells, however, results in their transport to the lysosomal compartment. Monitoring the cell surface appearance of the chimeric protein suggests that the majority is transported directly to the lysosomal compartment. Our results reflect cell type differences in the interpretation of the transmembrane domain Golgi retention signal and demonstrate that proteins which escape Golgi retention may be channeled to the lysosomal pathway.Thesis