Cellular Protein Transport and the Endoplasmic Reticulum

Protein Transport to the Endoplasmic Reticulum (ER)

Proteins secreted from a cell must be transported to the ER either during or after translation.

Function of Ribosomes: Ribosomes are responsible for protein synthesis (translation).

Initiates in the cytosol.
Translation pauses when a specific amino acid sequence is translated.
The translation complex is transported to the ER surface, where translation continues.

Proteins are fully translated in the cytosol.
Then, transported to the ER.
In both mechanisms, proteins enter the ER through a protein channel in the ER membrane.

Researchers used small interfering RNAs (siRNAs) to reduce the expression of SR or Sec62.
Cells were treated with either SR siRNA or Sec62 siRNA.
The relative amount of SR and Sec62 protein was determined and compared to cells treated with a control siRNA (Figure 1).

Shows average relative amounts of Sec62 and SR proteins in cells treated with control siRNA, Sec62 siRNA, or SR siRNA.
Error bars represent +SE (Standard Error).

The dependent variable is the relative protein content (% of control) of Sec62 and SR proteins.

The control (measuring both Sec62 and SR proteins in cells treated with Sec62 siRNA only) is crucial for comparison. It demonstrates the specificity of the siRNA effect: Sec62 siRNA should primarily reduce Sec62 protein levels, not SR protein levels, if the siRNA is working as expected. This ensures that any observed changes are due to the specific knockdown of Sec62.

Based on Figure 1, when cells are treated with Sec62 siRNA, there is a slight, likely insignificant, increase in the production of SR protein.

The researchers measured the amount of three different proteins transported to the ER in cells treated with Sec62 siRNA or SR siRNA.
Results were calculated as a percent transported relative to cells treated with control siRNA (Figure 2).

Shows average relative amounts of three proteins (Protein 1, Protein 2, Protein 3) transported to the ER when treated with control siRNA, Sec62 siRNA, or SR siRNA.
Error bars represent +SE.

The independent variable is the type of siRNA added to the cells (Control, Sec62 siRNA, or SR siRNA) for each protein.

Based on Figure 2, Protein 3 shows an increase in percent transport to the ER when treated with Sec62 siRNA compared to the control.

Protein 1 is encoded by 234 nucleotides.
Protein 2 is encoded by 495 nucleotides.
Since each codon (3 nucleotides) encodes one amino acid, we divide the number of nucleotides by 3 to find the number of amino acids.
Amino acids in Protein 1: \frac{234}{3} = 78
Amino acids in Protein 2: \frac{495}{3} = 165
Difference in the number of amino acids: 165 - 78 = 87

Researchers claim Protein 1 is transported to the ER following its complete translation in the cytosol (post-translational).
Supporting Data from Figure 2: When SR siRNA is applied, transport of Protein 1 to ER significantly decreases compared to control, and when Sec62 siRNA is applied transport of Protein 1 to ER slightly increases compared to control. This suggests that Protein 1 predominantly uses the Sec62-dependent (post-translational) pathway.

Researchers claim that amino acids near the protein's amino terminus determine how likely the protein is to pass through the protein channel within the ER membrane.
Justification: Signal sequences, often located near the amino terminus, are crucial for targeting proteins to the ER. These signal sequences interact with the protein channel, facilitating the protein's entry into the ER lumen. The characteristics (hydrophobicity, charge) of these amino-terminal amino acids influence the efficiency of this interaction and, thus, the likelihood of transport across the ER membrane.