Dartmouth Medical School’s Chair and Professor of Biochemistry Charles Barlowe
Dartmouth Medical School’s Chair and Professor of Biochemistry Charles Barlowe recently discovered that Ye1p and Ye3p yeast proteins interact with the ER translocation apparatus and are required for inositol prototrophy. The findings were recently published in Journal of Biological Chemistry.
BAP 29 and BAP 31, both B-cell receptor associated proteins, affect the function of the endoplasmic reticulum (ER), an organelle essential for protein synthesis, packaging, and secretion. Barlowe and his research team investigated these proteins by utilizing their yeast homologs, Yet1p and Yet3p. Among other findings, they determined that Yet1p and Yet3p form a complex, termed the Yet complex, which ensures correct localization of protein subunits in the ER.
Various biochemical techniques were utilized to perform the analysis, including PCR, a procedure that generates billions of copies of a given DNA sequence. Transformation was another important component. During transformation, PCR-amplified genes are inserted into a circular piece of DNA called a plasmid, which the investigators subsequently incorporated into the yeast genome. The transformed yeast can then be analyzed to determine how the gene in question affects the cell after it is translated and transcribed into protein.
The researcher team performed immunoblotting, a technique that employs antibodies to determine cellular protein expression. In this procedure, polyacrylamide gel electrophoresis separates proteins based on their size, and specific antibodies can be raised against the separated proteins to identify the relative sizes and amounts present.
This study provided several insights into the importance of Yet1p and Yet3p, and, by extension, their mammalian homologs in ER homeostasis. The data gathered indicates a link between the Yet complex and the ER translocation apparatus, or Sec complex. This interaction, moreover, is enhanced during times of ER stress. Further, they determined that Yet-Sec interactions increase when inositol, an alcohol that serves a structural role in eukaryotic secondary messengers, is absent.
Analysis of Yet mutants in the course of the investigation indicated, however, that the Yet proteins are not indispensable for the ER stress response. A triple Yet null mutant exhibited no growth-related problems unless the cells lacked inositol. The authors proposed that the removal of Yet1p and Yet3p has a deleterious effect on the translocation of proteins that respond to inositol shortages.
In biochemical terms, the authors concluded that Yet1p and Yet3p interact via an association of coiled-coil motifs. Coiled coil motifs, common components of protein tertiary structure, involve interactions between α-helices, polypeptide spirals stabilized by hydrogen bonds at regular intervals. These coiled-coil domains associate with the recognition domains of the Sec complex, allowing the Yet-Sec interactions the authors identified. Since the cytoplasmic coiled-coil motifs mediate the Yet-Sec association, Yet’s transmembrane domains are free to supervise the synthesis of translocation molecules.
The research team’s findings constitute an important step forward in the study of how the endoplasmic reticulum controls protein packaging and secretion. Further investigation will help establish further mechanisms of Yet1p and Yet3p action, which will in turn shed more light on their mammalian counterparts.