Consensus sequence information can provide valuable insights into the function o

Question

Consensus sequence information can provide valuable insights into the function of
an unknown protein. The sequences below represent the C-terminal region of a
highly conserved protein.

Based on your understanding of protein sequences:

Predict the organelle compartment that this protein most likely resides in
and explain your reasoning

How might you test if your prediction regarding location is true? Include the
following components in your answer.

Hypothesis: The protein belongs in the ___________________ compartment.
Experimental Control:
Experimental Group:
Method(s):
Result/Analysis:

What other type of sequences will you likely find on this protein beyond the
shown segment above based on your understanding of how this protein is
directed to the right compartment and how it may work?

Human .......FSKFLDZGGVLPTEESPPAAPFNSTDGSKDEL Rat .......FSKFEDNGAVLPTEESPPCAPFNSTMGSKDEL Cow ......FSQFLDZMVLPTEESPPAAKPFNSTMGSKDEL Mouse ......FSKFLDNGGVLPWEESPPAAMFNSTMGSKDEL

Explanation / Answer

Experimental Control: Choose a known protein like Bip (molecular chaperon present in ER). Clone the C-terminal region of Bip (also has KDEL sequence at the end) in a GFP expression vector.

Experimental Group:

Group1-Clone these regions (encoding this protein sequence) in a GFP expression vector separately.

Group2-Clone these regions (except mutation in the KDEL sequence) in a GFP expression vector separately.

Method(s): Now transfect the above constructs in a mammalian cell and check the expression. Check through live cell localization under a fluorescence microscope. Use ER tracker for marking of ER during microscopy.

Result/Analysis: Here, we have taken Bip as a positive control. It should show an ER localization and colocalize with ER tracker.

As all the above sequence also posses 'KDEL' sequence, they should show localization within ER and colocalize with ER tracker.

However, we also made some mutation in KDEL sequence. Now, these construct won't show an ER localization, suggesting that the KDEL sequence is active.

Note: During the construct preparation we should also consider putting an ER-targeting sequence at the N-terminus. Otherwise, these proteins can't enter ER. Remember the 'KDEL' sequence is only for ER retrieval not for ER targetting.

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