• Ph.D. 1971, University of Colorado
• Postdoctoral Fellow 1971-73, Massachusetts Institute of Technology
• Faculty Member, 1974-93, University of Wisconsin

Kenneth Keegstra

Research Interests

Plant cells contain two structures not found in animal cells: plastids and cell walls. Each plays an important role in the proper functioning of plants, and our laboratory is studying the biogenesis of both. For information on our studies on plant cell walls, performed jointly with Jonathan Walton's group, see Cell Wall Biosynthesis.

Plastid biogenesis requires the post-translational import of nuclear-encoded precursor proteins. Although the basic outline of this import process is now available, many important details are missing. Our laboratory is employing reverse genetic strategies to investigate the function of selected components of the protein import apparatus and biochemical strategies to investigate how they interact with precursor proteins.

As shown in Figure 1, most chloroplast-targeted proteins are synthesized as larger precursors containing a transit peptide that is necessary and sufficient for targeting the precursor into chloroplasts. The early stages of precursor interactions with the general import apparatus require nucleoside triphosphates, as shown in Figure 1. This energy is most likely used by the GTP-binding components in the outer envelope membrane (Fig. 2). Complete transport of precursors requires the hydrolysis of ATP inside chloroplasts (Figs. 1, 2). Based on current information, most, if not all, proteins destined for other compartments inside chloroplasts utilize the general import pathway. This is true even of proteins destined for the inner envelope membrane and the intermembrane space.

Figure 1. Schematic overview of the general import pathway. Precursor proteins containing an amino terminal transit peptide interact with proteins and lipids on the surface of chloroplasts. Formation of an early translocation intermediate requires low levels of nucleoside triphosphates, whereas complete translocation of precursors across both envelope membranes requires ATP hydrolysis in the stromal space. Either during or immediately after transport across the envelope membranes, proteins not destined for the stromal space are directed to their proper location, including the intermembrane space between the two envelope membranes (not shown), the inner envelope membrane, the thylakoid membrane, or the thylakoid lumen. IM, inner membrane; MC, molecular chaperone; NTP, nucleoside triphosphates; OM, outer membrane.

Biochemical strategies involving chemical cross-linking or solubilization of envelope membranes with mild detergents have led to the identification of a number of envelope membrane proteins that form the translocation apparatus in the outer and inner envelope membranes (Akita et al., 1997; Nielsen et al., 1997). Using variations of these two strategies, work from several laboratories has produced a relatively consistent picture of the identity of the transport components, shown schematically in Figure 2. One significant piece of missing information is the molecular details regarding the function of each component in the translocation apparatus. In many cases, the postulated function of each component is based on weak or circumstantial evidence. Thus, one major challenge for the future is to gain a more complete understanding of the function of each component, specifically, how each interacts with precursors and with other components of the transport apparatus to accomplish the movement of proteins across one or two biological membranes. One powerful strategy for addressing these functional questions is to use reverse genetics, as is now possible in Arabidopsis and other model systems.

Figure 2. Working model for transport across the two envelope membranes. Precursors are thought to interact reversibly with lipids and possibly some protein components, before a GTP-dependent step and an ATP-dependent step result in the formation of an early translocation intermediate (stage c). Precursors at this stage of import are thought to be engaged with some components of the inner envelope membrane, but are still sensitive to degradation by exogenous proteases. The precise functions of each component are still speculative and more experimental evidence is needed to evaluate this working model. IM, inner membrane; OM, outer membrane.

Our current efforts are focused on the components of the Tic complex, especially Tic40, Tic110, and Hsp93 (Chou et al., 2003; Constan et al., 2004b). We are especially interested in testing the hypothesis that Hsp93 provides the driving force that moves precursor proteins into chloroplasts.