Experimentally Assessing Protein Translocation

One way to assess the translocation of a given protein across the ER membrane and through subsequent vesicles would be by means of colocalization studies. Doing so requires an inducible protein of interest and a fluorescently tagged antibody specific to this protein. The protein must first be upregulated by inducing a promoter driving its expression, and cells should be briefly incubated before being fixed and permeabilized. These cells should then be exposed to fluorescently tagged antibodies of (coupled to two different fluors) specific to the protein of interest and to an ER-resident marker protein such as calnexin or calreticulin. Cells should then be imaged using immunofluorescence microscopy at the excitation/emission wavelengths corresponding to the two fluorescently tagged antibodies, and the two resultant images can be digitally overlayed on one another. If the fluorescence patterns of the two images overlap heavily and specifically then this indicates the colocalization of these two proteins, indicating that the protein of interest has entered the ER lumen.

An alternative means of establishing translocation of the ER membrane involves an in vitro translation system, as previously described (Waters, 1986). The mRNA coding for the protein of interest must first be isolated and amplified, and must then be transcribed in vitro in cytoplasm + microsomal membranes extracted from WT cells of the species of interest. After this mixture has been allowed to incubate for some time, trypsin should be added with or without Triton X-100 in order to digest exposed proteins. After a short incubation, trypsin activity should be quenched using phenylmethylsulfonyl fluoride (PMSF, a protease inhibitor) and the solution should be analyzed by Western blot to test for the presence of the protein of interest. If the protein is present in samples exposed to trypsin but not Triton X-100 then this indicates that the protein was protected from proteolysis due to its incorporation into microsomal vesicles, demonstrating the ER translocation of the protein.

A third technique for assessing ER localization relies upon pulse-chase labeling of an inducible protein, similar to the techniques of Siekevitz + Palade (Siekevitz, 1960). Briefly, the gene encoding the target protein must be put under the control of a selectively inducible promoter which must be induced at the same time as a radiolabeled amino acid is added. After a brief labeling period, the labeled amino acid must be washed out. At various time points thereafter, cells should undergo high speed centrifugation to isolate individual organelle populations and run on SDS-PAGE gels, and the radioactivity of bands corresponding to the protein of interest in each organelle fraction should be assessed. Doing so should allow for the establishment of a stepwise progression of the protein from its translocation of the ER membrane to its ultimate destination.

The outcome of these experimental procedures should not vary significantly between proteins of interest which are soluble secreted proteins when compared with proteins which contain internal stop transfer/signal anchor sequences and ultimately become transmembrane proteins. Both classes of protein should produce the same results during pulse-chase labeling and colocalization experiments. During the protease protection assay the cytoplasmic domains of transmembrane proteins will be cleaved by the trypsin resulting in a lower molecular weight species when a Western blot is performed, but so long as this cleavage doesn’t disrupt the antibody binding epitope this should not affect the ultimate interpretation of the results.

Experimentally Determining the Time of Translocation

In order to establish whether a novel protein is translocated into the ER co- or post-translationally, there are numerous experimental techniques which may be employed. Both methods of translocation involve the Sec61 protein, however the translation of co-translationally translocated proteins is arrested by SRP until the SRP-ribosome complex binds to the ER. As a result, if the novel protein were post- but not co-translationally translocated it should be present at low levels in the cytoplasm. Using a Sec61-ts yeast strain which does not permit significant translocation at non-permissive temperatures, it would be possible to accumulate detectable levels a post-translationally translocated protein in the cytoplasm due to the nonfunctional Sec61 protein. Accordingly, the cytoplasmic fraction of Sec61-ts yeast should be obtained by centrifugation and assayed via Western blot using an antisera against the novel protein in order to assess whether the protein is present in the cytoplasm at higher levels when Sec61 is non-functional than when it is functional. Elevated cytoplasmic levels of the novel protein would suggest post-translational translocation as its primary mechanism of ER localization.

Another experimental technique to assess means of ER translocation would be to assess whether the protein can be post-translationally translocated in a cell free system using a modification of the aforementioned protease protection assay previously described by Waters + Blobel (Waters, 1986). First, the mRNA encoding the novel protein must be isolated and amplified via PCR. Next, the mRNA must be translated in vitro in the absence of microsomal membranes using appropriate compounds (“master mix” + “energy mix” in Waters + Blobel paper). RNAse should then be added to prevent further translation, and WT-yeast microsomal membranes should then be added and the mixture should be incubated for a short period. Trypsin should then be added in the presence or absence of Triton X-100. The digestion can then be terminated using PMSF and the samples can be analyzed using a Western blot. If the protein was protected from digestion in the absence of Triton X-100 then this indicates that the protein was post-translationally translocated into the added microsomes.

A final set of experiments hinges upon the requirement of SRP and SRP-receptor activity for co- but not post-translational translocation to occur. In order to test whether or not SRP arrests translation, purified mRNA of the novel protein should be translated in vitro without microsomes in the presence or absence of SRP. The proteins in solution should then be assessed by Western blot in order to establish whether or not the addition of SRP arrested translation as it would for a co-translationally translocated protein. If so, microsomes should be added in order to assess whether or not this alleviates translational inhibition by SRP; if it does then this further strengthens the argument that this is a co-translationally translocated protein. Expression of the protein of interest can also be examined in a yeast strain with a temperature sensitive mutation for the beta subunit of the SRP-receptor which significantly limits co-translational translocation at non-permissive temperatures (Ogg, 1998). If this protein can be detected via Western blot of whole cell lysate at permissive but not at non-permissive temperatures, then this protein likely undergoes co-translational translocation. This effect would likely be significantly enhanced if the cells were treated with tunicamycin to prevent glycosylation within the ER, causing proteins to accumulate and enhancing the signal of the target novel protein.

These techniques would produce the same results regardless of whether the protein of interest was a secreted or a transmembrane protein with the exception of the protease protection experiment. If the protein had a cytosolic domain then this domain would be cleaved by trypsin, resulting in a protein of lower molecular weight during subsequent Western blot analysis. While the molecular weight of the protein will have shifted, the ability to draw conclusions as to whether or not the protein is post-translationally translocated will remain the same so long as the antibody binding epitope of the protein was not cleaved.


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