Analyze The Data

Mouse liver cells were homogenized and the homogenate subjected to equilibrium density-gradient centrifugation with sucrose gradients. Fractions obtained from these gradients were assayed for marker molecules (i.e., molecules that are limited to specific organelles). The results of these assays are shown in the figure. The marker molecules have the following functions: Cytochrome oxidase is an enzyme involved in the process by which ATP is formed in the complete aerobic degradation of glucose or fatty acids; ribosomal RNA forms part of the protein-synthesizing ribosomes; catalase catalyzes decomposition of hydrogen peroxide; acid phosphatase hydrolyzes monophosphoric esters at acid pH; cytidylyl transferase is involved in phospholipid biosynthesis; and amino acid permease aids in transport of amino acids across membranes.

peripheral membrane protein 157 peroxisome 168 phosphoglyceride 150 phospholipid bilayer 149 pleckstrin homology (PH)

domain 163 porin 160 prenyl anchor 160 resolution 184 sphingolipid 151

Fraction number 50% <-Sucrose 0%

Curve A = cytochrome oxidase Curve D = acid phosphatase Curve B = ribosomal RNA Curve E = cytidylyl transferase

Curve C = catalase Curve F = amino acid permease a. Name the marker molecule and give the number of the fraction that is most enriched for each of the following: lysosomes; peroxisomes; mitochondria; plasma membrane; rough endoplasmic reticulum; smooth endoplasmic reticulum.

b. Is the rough endoplasmic reticulum more or less dense than the smooth endoplasmic reticulum? Why?

c. Describe an alternative approach by which you could identify which fraction was enriched for which organelle.

d. How would addition of a detergent to the homogenate affect the equilibrium density-gradient results?

Sprong, H., P. van der Sluijs, and G. van Meer. 2001. How proteins move lipids and lipids move proteins. Nature Rev. Mol. Cell Biol. 2:504-513.

Tamm, L. K., V. K. Kiessling, and M. L. Wagner. 2001. Membrane dynamics. Encyclopedia of Life Sciences. Nature Publishing Group.

Vance, D. E., and J. E. Vance. 2002. Biochemistry of Lipids, Lipoproteins, and Membranes, 4th ed. Elsevier.

Yeager, P. L. 2001. Lipids. Encyclopedia of Life Sciences. Nature Publishing Group.

Biomembranes: Protein Components and Basic Functions

Cullen, P. J., G. E. Cozier, G. Banting, and H. Mellor. 2001. Modular phosphoinositide-binding domains: their role in signalling and membrane trafficking. Curr. Biol. 11:R882-R893.

Lanyi, J. K., and H. Luecke. 2001. Bacteriorhodopsin. Curr. Opin. Struc. Biol. 11:415-519.

MacKenzie, K. R., J. H. Prestegard, and D. M. Engelman. 1997. A transmembrane helix dimer: structure and implications. Science 276:131-133.

Minor, D. L. 2001. Potassium channels: life in the post-structural world. Curr Opin. Struc. Biol. 11:408-414.

Schulz, G. E. 2000. ß-Barrel membrane proteins. Curr. Opin. Struc. Biol. 10:443-447.

Organelles of the Eukaryotic Cell

Bainton, D. 1981. The discovery of lysosomes. J. Cell Biol. 91:66s-76s.

Cuervo, A. M., and J. F. Dice. 1998. Lysosomes: a meeting point of proteins, chaperones, and proteases. J. Mol. Med. 76:6-12.

de Duve, C. 1996. The peroxisome in retrospect. Ann. NY Acad. Sci. 804:1-10.

Holtzman, E. 1989. Lysosomes. Plenum Press. Lamond, A., and W. Earnshaw. 1998. Structure and function in the nucleus. Science 280:547-553.

Masters, C., and D. Crane. 1996. Recent developments in per-oxisome biology. Endeavour 20:68-73.

Palade, G. 1975. Intracellular aspects of the process of protein synthesis. Science 189:347-358. The Nobel Prize lecture of a pioneer in the study of cellular organelles. (See also de Duve, 1996.)

Subramani, S. 1998. Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement. Physiol. Rev. 78:171-188.

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