The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34؉ erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61␣), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.

Mutations in the human kidney anion exchanger 1 (kAE1) membrane glycoprotein cause impaired urine acidification resulting in distal renal tubular acidosis (dRTA). Dominant and recessive dRTA kAE1 mutants exhibit distinct trafficking defects with retention in the endoplasmic reticulum (ER), Golgi, or mislocalization to the apical membrane in polarized epithelial cells. We examined the interaction of kAE1 with the quality control system responsible for the folding of membrane glycoproteins and the retention and degradation of misfolded mutants. Using small molecule inhibitors to disrupt chaperone interactions, two functional, dominant kAE1 mutants (R589H and R901stop), retained in the ER and targeted to the proteasome for degradation by ubiquitination, were rescued to the basolateral membrane of Madin-Darby canine kidney cells. In contrast, the Golgi-localized, recessive G701D and the severely misfolded, ER-retained dominant Southeast Asian ovalocytosis (SAO) mutants were not rescued. These results show that functional dRTA mutants are retained in the ER due to their interaction with molecular chaperones, particularly calnexin, and that disruption of these interactions can promote their escape from the ER and cell surface rescue.

SUMMARYThe structure of mouse submaxillary gland epidermal growth factor (EGF) precursor has been deduced from complementary DNAs. The mRNA is approximately 4800 bases and predicts prepro EGF to be a protein of 1217 amino acid residues (133X 10M r). EGF (53 amino acid residues) is flanked by polypeptides of 188 and 976 residues at its carboxy and amino termini, respectively. The amino terminus of the precursor contains seven cysteine-rich peptides that resemble EGF. Towards the carboxy terminus is a 20-residue hydrophobic membrane spanning domain. The mid portion of the EGF precursor shares a 33 % homology with the low density lipoprotein receptor, which extends over 400 amino acid residues. These features suggest that EGF precursor could function as a membrane-bound receptor.RNA dot-blot analysis and in situ hybridization show EGF mRNA to be abundant in the submaxillary gland, kidney and incisor tooth buds. Lower EGF mRNA levels were found in the lactating breast, pancreas, small intestine, ovary, spleen, lung, pituitary and liver. In the kidney EGF mRNA was most abundant in the distal convoluted tubules. Analysis of EGF precursor biosynthesis in organ culture of the submaxillary gland and kidney showed differential processing of the precursor in the two tissues. In the submaxillary gland immunoreactive low molecular weight EGF was produced, but in the kidney the high molecular weight precursor was not processed. In the distal convoluted tubule of the kidney EGF precursor may act as a receptor that is involved in ion transport.

Metabolism, including cellular respiration, encompasses a large component of the topics covered in foundational biochemistry and life science undergraduate courses. The difficulty in instruction and student retention of this topic lies in the conceptualization of the numerous enzymatic reactions involving various substrates/products and their regulation. Learners typically end up focusing on memorizing individual metabolic pathways and their subcomponents (e.g., enzymes, substrates, and products) rather than developing a high-level understanding of the interplay and regulation occurring between pathways and appreciating the real-life application of this knowledge. To address this challenge, we created a 3D animation that introduces students to the fundamental concepts of glucose and fat metabolism, energy production by the citric acid cycle and oxidative phosphorylation, as well as the major points of regulation that emphasize the integration and flux of these metabolic pathways. Survey feedback from cohorts of University of Toronto second-year undergraduate life science students and School of Continuing Studies learners indicated that the animation increased their engagement with the material and confidence in understanding the connectivity between competing pathways and the regulation of metabolism.

The presence of iron on the transferrin molecule increases its affinity for and sojourn time on the reticulocyte. This could be due to selective internalization of iron-containing transferrin molecules. This possibility was investigated by electron microscopic autoradiography. Rabbit reticulocytes were incubated with rabbit transferrin at 6, 33, and 72% iron saturations, and the distribution of transferrin molecules at membrane and intra-cellular locations was assessed by grain counting. The results showed that (1) both apo-transferrin and iron transferrin enter the cell interior and (2) the amount of intracellular transferrin was primarily controlled by the concentration of membrane-bound transferrin and not by its iron saturation.