The examination of cells by light and electron microscopy led to the appreciation that eukaryotic cells contain a common set of organelles, introduced in Chapter 1 (see Figure 1-12a). However, observing organelles and documenting their detailed structure by microscopy does not clearly reveal the roles they play and how they work. For this, it is necessary to isolate organelles in their native state and identify and dissect the function of each component. For this reason, methods to isolate and characterize organelles were developed in parallel with advances in microscopy. Lysosomes, for example, are organelles in which biological molecules are degraded, as described in Chapter 1. Lysosomes had been seen by microscopy, but their function was discovered only after a method was developed to isolate them. When a method is developed to purify a type of organelle, it is possible to begin to catalogue all of its components and probe the function of each one. In another example, electron microscopy revealed that some ribosomes are associated with the endoplasmic reticulum, suggesting that the ER is a site of protein synthesis. We now know that proteins to be secreted from the cell are made by these ribosomes, and that the nascent secretory protein is translocated across the membrane into the lumen of the endoplasmic reticulum. As we describe in Chapter 13, understanding the mechanism by which this occurs depended on the isolation of the endoplasmic reticulum and the development of in vitro assays for the synthesis and translocation of secretory proteins. Thus, before one can begin to fully understand organelles, biochemical assays need to be established to probe the functions of each component, with the eventual goal of reconstituting functional organelles from purified components.

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Most organelles are enclosed in a lipid bilayer and perform a specific function. Each type of organelle has a recognizable structure and contains a specific set of proteins to perform its function. Cell biologists use this fact to identify specific organelles. For example, as discussed in Chapter 12, most of the ATP in a cell is made by ATP synthase, which converts ADP to ATP in mitochondria, so ATP synthase is a good marker for mitochondria. As we will discuss below, the availability of specific markers for organelles has helped in the development of organelle purification.

In this section, we discuss methods that are used to open up cells for the purification of organelles. We end with recent advances in proteomics aimed at defining the complete protein inventories of organelles.