Investigating the Function of a Transport Protein:
Where is ABCB6 Located in Human Cells?
Instructions:
1) Take a moment to review your assigned role: manager, recorder or presenter.
2) As a group, answer the questions below in the order they appear.
3) Your recorder should turn in one copy for your group with everyone’s name on it.
Learning Objectives:
1) Be able to describe the use of two common research techniques for studying proteins: SDS-PAGE and immunoblot analysis.
2) Be able to determine a protein’s subcellular location based on results from: 1) immunoblotting after differential centrifugation, and 2) immunofluorescence microscopy.
3) Be able to analyze protein localization data based on the limitations of differential centrifugation and immunofluorescence microscopy.
Role / Name / ID NumberManager
Recorder
Presenter
Important Background
Cell biologists investigate the functions of cellular proteins in order to better understand cell function. Identifying the location of a protein is an important first step toward understanding its function. For example, Katalin Kiss studied a transport protein from the adenosine triphosphate-binding cassette (ABC) family called ABCB6. Most human ABC proteins are used to pump substance out of cells or out of organelles, in a process known as efflux.
Researchers in other labs predicted that ABCB6 is located in mitochondrial membranes, and they predicted that ABCB6 is responsible for porphyrin uptake into mitochondria. Kiss and her colleagues questioned the data to support these predictions. They wanted to investigate ABCB6 themselves, and they started by asking the following research question: Where is ABCB6 located in human cells? In this problem set, we will explore some of the data that Kiss and her colleagues used to identify the subcellular localization of ABCB6.
Kiss K, Brozik A, Kucsma N, Toth A, Gera M, Berry L, Vallentin A, Vial H, Vidal M, Szakacs G. 2012. Shifting the Paradigm: The Putative Mitochondrial Protein ABCB6 Resides in the Lysosomes of Cells and in the Plasma Membrane of Erythrocytes. PLoS One. 7 (5): e37378.
1) Kiss and her colleagues used some common techniques for studying proteins to identify the location of ABCB6. For this first question, you will review two techniques you have covered in previous courses: SDS-PAGE and immunoblot analysis.
a) What is SDS-PAGE used for and how does it work? You can use your computer to look up SDS-PAGE if needed.
b) Above is a figure showing the key steps for immunoblot analysis (also known as a Western blotting). What is an immunoblot used for and how does it work?
c) Why would a scientist choose immunoblot analysis over a SDS-PAGE gel alone?
2) One way to localize a protein is to separate cell components into fractions and test whether or not a protein is found in the same fractions as marker proteins specific for organelles. For example, porin is a marker protein for mitochondria because it is only found in mitochondrial membranes. Differential centrifugation can be used to separate cell components based on size and density using increasingly faster centrifuge spins. The larger the component, the less centrifugal force you need to pellet the component.
First, cells must be broken open or lysed, which results in a whole cell lysate. Next, the lysate is centrifuged at a low speed. The pellet of the low-speed spin usually contains nuclei, which is the largest organelle in the cell. The supernatant of the low-speed spin usually contains all other cell components. Next, the supernatant of the low-speed spin is centrifuged at a medium speed. The pellet of the medium-speed spin may contain mitochondria and chloroplasts. The centrifugation step is repeated with the supernatant from the previous spin centrifuged at a higher speed. A very high-speed spin can pellet very small cell components, such as ribosomes.
a) A hypotonic solution can be used to help lyse cells. Briefly explain how a hypotonic solution could break cells open.
3) Kiss and her colleagues used differential centrifugation to separate cell components. They lysed human cells to obtain a whole cell lysate (lane 1). Then they used the whole cell lysate in differential centrifugation and obtained four pellets from four different centrifugation steps (lanes 2-5 below). They separated the proteins in the pellets on an SDS-PAGE gel and performed four separate immunoblots to locate marker proteins (porin, NaK-ATPase, or LAMP1) or ABCB6.
In this space, please insert Figure 1A,which can be freely downloaded from:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037378
Figure 1A contains immunoblots after differential centrifugation to identify location of ABCB6 transporter. / Lane 1: whole cell lysate
Lane 2: 1,300 x g pellet
Lane 3: 8,000 x g pellet
Lane 4: 12,000 x g pellet
Lane 5: 20,000 x g pellet
Note: Each panel is a separate
immunoblot for a different
protein.
a) Where could an ABC transporter be found in a cell? You can use the internet to look this up.
b) Explain why porin, NaK-ATPase, and LAMP1 are good marker proteins for the mitochondria, plasma membrane, and lysosomes, respectively. You can use your computer to look this up.
c) Describe the data you see in the figure above. Where do you think ABCB6 is found in K562 cells? Explain your answer.
d) Did the researchers achieve a high level of organelle purity in any of their pellets? How can you tell?
4) Another way to test the location of a protein is to use fluorescence microscopy. Similar to immunoblot, immunofluorescence involves the use of a primary antibody that specifically recognizes a protein of interest. Then a secondary antibody linked to a fluorescent probe is used to recognize the primary antibody so that the location of the protein can be visualized under a microscope. Kiss and her colleagues used this approach to further examine the location of ABCB6 (green) as compared to marker proteins (red) in human cells. Yellow is seen when the red and green overlap.
In this space, please download and insert Figure 2A,which can be freely downloaded from:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037378
Figure 2A contains immunofluorescence assay micrographs to identify location of ABCB6 transporter. / CoxIV (mitochondria)
Lamp1 (lysosomes)
Each panel only
shows one cell.
a) What organelle is blue in every panel? Hint: What organelle is likely to be of the size and shape of the blue organelle?
b) Describe the data you see in the figure above. Where do you think ABCB6 is found in human cells?
c) Can immunofluorescence definitively tell you the location of a protein in cell? Explain your answer. Hint: Remember that you are viewing 2D images of 3D cells.
d) Why do you think the researchers used more than one approach to test the location of ABCB6?
5) Why do you think Kiss and her colleagues were interested in the subcellular localization of ABCB6? Why does it matter where a protein is located in the cell?
Copyright: The authors affirm that they have written permission to use the text, figures, tables, artwork, abstract, summaries and supplementary materials, or they are using these items under a Creative Commons license.
The figures shown in Questions 1 and 2 (SDS-PAGE and immunoblotting figure and differential centrifugation figure) were commissioned from a graphic artist, Ashley Rasys of Athens, GA. Ms. Rasys signed over all rights to use and publish the figures to the authors (Stanton & Dye).
The data for Questions 3 and 4 (immunoblot data and immunofluorescence data) can be freely obtained from the Kiss et al., 2012 PLoS One paper, which was published under a Creative Commons license. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037378
Investigating the Function of a Transport Protein: Where is ABCB6 Located in Human Cells?
Julie Dangremond Stanton & Kathryn Morris Dye