Question

Dear anon Yes it is, does that mean I can't ask questions??

Problem 4.1.6 (tags: #gel electrophoresis, #drug treatments, #protein glycosylation) In vitro protein synthesizing systems, containing free amino acids, mRNA, and cytosolic extracts, can be used to study protein synthesis. The cytosolic extracts provide ribosomes and all cellular proteins and factors necessary for protein synthesis. The gel on the right is a protein gel showing the results of experiments conducted using this in vitro system, the mRNA of a glycoprotein (protein X) and the following experimental conditions: Lane 1: protein X was synthesized in the absence of ER membrane preparations or tunicamycin (see notes below). Translation of Protein X in the presence of: Tunicamycin ER Membrane Lane 2: protein X was synthesized in the presence of ER membrane preparations in the medium and in the absence of tunicamycin. 28 kDa 26 kDa Lane 3: protein X was synthesized in the presence of both ER membrane preparations and tunicamycin. Notes: 24 kDa 1. For the experimental samples analyzed in Lanes 2 1 2 3 and 3, all co-factors necessary for co- translational transport are provided with the ER membrane preparations. 2. Tunicamycin is an inhibitor of glycosylation and can cross the ER membrane. Questions: a) Describe the data for each treatment. b) Explain the differences in lane 1 versus lane 2. c) Explain the differences in lanes 1 and 2 versus lane 3.

Answer 1

Lane 1 shows that protein formed without post-transcriptional is of 26kDa.

Lane2 shows that protein formed in the presence of ER membrane i.e after posttranslational modification it is of 26kDa.

Lane 3 shows that protein is amid of posttranslational modification i.e protein has entered the lumen and membrane of RER by the process of co-translational translocation. There N-linked glycosylation of proteins take place for proper folding and functioning. Addition of Tunicamycin blocks the first step of N-linked glycosylation and thus inhibit the synthesis of oligosaccharide.

B) lane 1 represents the protein formed just after translation , lane 2 shows protein formed after post-translational modification.

C) lane 1 shows protein without post translational modification and lane2 shows a protein that is amid of posttranslational modification , lane3 shows protein formed after complete post-translational modification.