Protein Import and Routing Systems of Chloroplasts
Year: 1999
Type: article
Abstract: Plastids are ubiquitous plant cell organelles that perform many essential functions. Chloroplasts are the most complex type of plastid, both structurally and functionally, containing six distinct compartments and performing essential processes, such as fatty acid and amino acid biosynthesis in addition to their well-known role in photosynthesis. The many plastid proteins needed for these and other essential metabolic pathways derive from two genetic systems. The plastid genome encodes ∼100 proteins (Sugiura, 1989), but the vast majority of plastid proteins are encoded by nuclear genes and synthesized as precursors in the cytoplasm. Consequently, specific and reliable protein transport mechanisms are needed to direct these precursor proteins to their proper location within chloroplasts. Most studies of protein transport to and within chloroplasts have been performed with isolated organelles, whereby the general process of protein transport, as schematically presented in Figure 1, can be broken down into three distinct phases: specific targeting of precursor proteins to plastids, transport across the two envelope membranes, and in many cases, sorting to the proper plastidic compartment. One major conclusion derived from these studies is that proper localization of cytoplasmically synthesized chloroplastic proteins is accomplished by specific interactions between targeting sequences contained within the precursor proteins and the transport machinery contained within chloroplasts. This review focuses on the transport process and the machinery that mediates it; it also includes a brief consideration of targeting sequences. Many excellent reviews have covered earlier progress in understanding chloroplastic protein transport (Keegstra et al., 1989; de Boer and Weisbeek, 1991; Schnell, 1995, 1998; Cline and Henry, 1996; Kouranov and Schnell, 1996; Fuks and Schnell, 1997; Lübeck et al., 1997a; Heins et al., 1998); these reviews can be consulted for additional details. Most precursor proteins are larger than their corresponding plastid-localized forms. The N-terminal transit peptide that is cleaved off the precursor protein upon entry into chloroplasts (Figure 1) usually contains stromal-targeting information that is necessary and sufficient for transport of precursors across the two envelope membranes (Keegstra et al., 1989; de Boer and Weisbeek, 1991). In some cases, the cleavable transit peptide is bipartite and contains additional targeting information, as is best exemplified by proteins destined for the thylakoid lumen. In addition to the stromal-targeting domain, bipartite peptides of lumenal proteins contain a second domain that directs transport across the thylakoid membrane (Figure 1; de Boer and Weisbeek, 1991; Cline and Henry, 1996). The two domains of these bipartite transit peptides are removed by two separate processing proteases (Figure 1, path 3): one in the stroma (VanderVere et al., 1995), and one in the thylakoid lumen (Chaal et al., 1998). Many proteins, especially those destined for the inner envelope membrane or the thylakoid membrane, have both a cleavable stromal-targeting domain and additional targeting information retained within the mature protein (Figure 1, paths 2 and 4). A few precursors, especially those destined for the outer envelope membrane, do not have cleavable targeting sequences (Figure 1, path 6). In the latter case, information directing the protein to its proper location is contained within the mature protein (Cline and Henry, 1996). Despite their common function, stromal-targeting domains from various precursor proteins may vary in length from 30 to >100 amino acids. Interestingly, they share minimal sequence similarity so that no consensus targeting sequences have been established, and efforts to identify common secondary structures have not been successful (Keegstra et al., 1989; von Heijne et al., 1989; von Heijne and Nishikawa, 1991). Most stromal-targeting domains are rich in serine and threonine but deficient in acidic amino acids (Keegstra et al., 1989). The lumenal-targeting domain from bipartite transit peptides has strong similarity to the hydrophobic signal sequences found in secreted proteins (Cline and Henry, 1996). The targeting information contained within mature proteins generally is located in membrane-spanning domains, where it also can serve as a membrane insertion or a stop-transfer signal (Cline and Henry, 1996). Schematic Representation of Pathways Responsible for Targeting Proteins to Their Proper Location within Chloroplasts. The large majority of precursor proteins have a cleavable transit peptide (shown in red). In most cases, the transit peptide includes a stromal-targeting domain that initiates transport of the precursor through the general import pathway (shown in gray). The driving force for protein translocation is thought to be provided by molecular chaperones (MC) that pull precursor proteins into plastids. Precursors lacking additional targeting information are thus deposited into the stromal space, where the stromal-processing protease removes the transit peptide (path 1). For precursors destined for insertion into membranes, the additional targeting information generally is contained within the mature region of the protein (paths 2 and 4), although some proteins may require a stop-transfer signal for localization to the outer membrane (path 5). Precursors for some outer membrane proteins lack a cleavable transit peptide and are inserted directly into the outer membrane without using the general import pathway (path 6). Some precursors destined for the thylakoid lumen require a second targeting signal (shown in purple), which is cleaved as the proteins enter the lumen (path 3). See text for additional details. Transport across the two envelope membranes, mediated by stromal-targeting sequences, is a common step for all proteins that enter plastids, regardless of their ultimate destination in the organelles. Most of the proteins studied to date are imported via a single transport system, which is sometimes referred to as the general import pathway (Cline and Henry, 1996; Heins et al., 1998). Although most of the work so far performed has focused on this pathway, it is possible, even likely, that other routes exist for entry into chloroplasts. As detailed in Figure 2, transport across the envelope membranes can be divided into at least three stages. In the first stage, precursors associate reversibly with plastids (Figure 2, stage a). This interaction is thought to be mediated in part by protein–protein interactions and in part by protein–lipid interactions. Transit peptides interact specifically with lipids from the outer envelope membrane (Pinnaduwage and Bruce, 1996), and mutants deficient in galactolipids are impaired in their ability to import precursors (Chen and Li, 1998). One of for protein–protein interactions from studies that are in The two in import usually are by their of chloroplasts with precursor proteins in the of or of in a between precursors and the import but it the precursors in a in which they can be by proteases and This has been but is as translocation (Figure 2, stage of are translocation of precursors and the (Figure 2, stage The for translocation of precursors is within the et al., 1989), by molecular chaperones that pull precursors into the In to where a force is for protein import et al., a membrane is not needed for transport across the chloroplastic envelope membranes et al., 1989). progress has been in of the transport by of two and of with Although of these two have been by the generally of the translocation schematically in Figure The into three outer membrane proteins, inner membrane proteins, and molecular is outer envelope membrane proteins have been as part of the and is for at the outer membrane of the the molecular of the protein in et for three proteins interact with other in the outer envelope membrane to a complex even in the of that are in the process of translocation et al., 1996; et al., the of within the complex and the of to to in the is a protein that is to as import et al., and Although it membrane-spanning it is membrane by the et al., et al., is chloroplasts are to protease that it is on the chloroplastic that is the of Schnell, the of the is the one isolated envelope membrane proteins are by but is to a also is the isolated not the a protein of Schnell, the that the that encodes the is on the and is to a protein of The conclusion that is a derived from a larger protein has for studies on the of protein The to which is to may vary in of upon the to organelles. it is this the of the to be also a has sequence similarity to the N-terminal region of et al., is to have a single membrane-spanning its with the majority of its domain at the of the outer membrane (Chen and Schnell, 1997; and studies have that is also in to destined for but in the translocation process and Schnell, of or can that of to on this it that is also a for part of the complex (Figure Schematic Representation of the and in the of Precursors across and the in the translocation process are presented in the Although the and one of the of in the is not The of may be with or the of as is for and a second protein with sequence similarity to in et al., 1998). The that of the in protein both in and in the first in on the of the envelope translocation Interestingly, of the not of the of a in a is and is at The of the two genes and their is not and it is not other plant also have two distinct of this import the most protein in the outer envelope membrane, is to the through which precursor proteins are across the outer membrane et al., et al., is in the membrane and is to from the outer et al., it membrane-spanning and is to the membrane with The best its as a is from that in is of a in a membrane et al., many as a in the plastid outer envelope For is and in the outer envelope membrane, which a membrane other to be the of in Although most of the translocation of plastids lack sequence similarity to transport found in the membrane or the transport has sequence similarity to from et al., the which that chloroplasts from it is to the into the of the protein transport The of the protein to be established, but one is that it part of a by to proteins and that it for in the protein import et al., proteins have been as of the complex at the inner membrane of The best studied is which has been by two and 1996; Lübeck et al., 1996). Although both that has a membrane the they to its in the Lübeck et that the large domain into the space, and that it into the et this and that the domain is in the Although the of and that the stromal domain with molecular specifically studies do not of with they with the interaction that may have for the of import et al., 1997; et al., three other proteins have been as inner membrane translocation is found with other translocation by et al., This protein is translocation in that it contains The role for a in protein translocation to be it be that not found in with and or in the by Kouranov et and have been by a that both are with translocation and Schnell, is to be membrane part of the inner membrane translocation is a membrane protein and to inner membrane to outer membrane et al., 1998). a protein has been as part of the translocation machinery et al., the role of this protein is it has been to be a of both the inner and outer envelope chaperones essential in protein translocation into et al., and transport into the lumen of the and and in this is that they also are in chloroplastic protein and chloroplasts have molecular chaperones that are for a protein found on the of the outer envelope membrane, has been to a role in the of protein import and although other have not this as a of the complex et al., A located on the inner of the outer envelope membrane, has been as part of with (Figure 2, stage et al., This may be for with the outer envelope membrane et al., et al., In this it may the driving force for transport across the outer envelope membrane and may for the between transport across the outer envelope membrane and those across the inner membrane, as by and chaperones in the stromal are thought to protein import into the stroma by into chloroplasts through of and to the protein import of et al., a stromal be to chloroplastic protein no stromal be found in the et al., the contains a stromal protein of the of molecular chaperones et al., 1997; et al., work is needed to this is to the or it in with the stromal in that to be In this it is to that two separate have been for protein driving protein translocation and in the of et al., is that and to these two in chloroplasts. Despite progress in the of translocation to be their and they interact with other and with precursor proteins to protein have not been for and for and where have been the these least two pathways have been for targeting proteins to the outer envelope membrane (Figure and it is that The first (Figure 1, path is for the transport of various proteins, such as that are not synthesized with a cleavable N-terminal peptide et al., 1991; and Schnell, 1997; and of these proteins into the outer membrane of chloroplasts not require of it to on proteins of the outer that the into the of hydrophobic domains contained within these proteins their insertion into the membrane and 1996). A second pathway (Figure 1, path is by the which contains a bipartite transit peptide and 1996). The N-terminal stromal-targeting of the transit peptide the of the general import pathway and is removed by the stromal-processing The containing the second of the bipartite transit can be by in import and in in et al., is that the second domain of the transit peptide as a stop-transfer signal to translocation within the envelope membrane its mature and 1996). The second of the bipartite transit peptide is removed by Proteins destined for the inner envelope membrane have a transit peptide that of a stromal-targeting domain (Figure 1, path et al., et al., The information that directs these proteins to the inner envelope membrane, is contained within the mature region of the protein and has been by studies to hydrophobic domains et al., and Lübeck et al., to insertion of the imported protein from the stroma into the inner envelope a has been for localization et al., hydrophobic within proteins may as stop-transfer sequences to from the import machinery at the inner envelope that and to the inner membrane to in and et al., of which pathway is in the as to for envelope is Many thylakoid proteins contain hydrophobic that stop-transfer the other hydrophobic domains of thylakoid proteins as membrane insertion for thylakoid many the insertion pathway and membrane to be proteins are imported into the stroma by the general import machinery and are into or across the thylakoid membrane in Cline and Henry, 1996; and 1997; Schnell, 1998). a process is by of the of stromal and in for translocation into isolated The of such in which can be so as to the stromal proteins, and so has to into the protein import One of studies into the transport of proteins into is that precursor proteins into at least distinct on their 1). of precursor proteins translocation of transport by A of thylakoid proteins, all membrane proteins, and protein the of a insertion and for that distinct mechanisms are in the targeting translocation studies with precursor proteins in (Cline et al., the conclusion that thylakoid proteins are specifically to distinct translocation pathways their import into the stroma (Figure 3). As it generally is that chloroplasts from a to a it that thylakoid protein transport pathways be to machinery that proteins from the cytoplasm. In addition to the work of et of this from and genetic studies that thylakoid translocation are with those of the a between one thylakoid transport pathway and the system, which of translocation and a membrane to of membrane insertion and so as to of precursor proteins across the part of the translocation 1998). Transport of the of the complex and of to et al., and the that a protein be in thylakoid in Cline and and and a for the no a is membrane but the lumen. and A for not for in Cline and and and a for the no a is membrane but the lumen. and A for not for A from stroma and to transport of and into has no on the transport of and or on the of protein et al., a for and to transport of but not et al., of et al., and and also have been and to to a peptide transport of the precursors and the that in with (Figure 3). Although work to be that the of the pathway is to that of the is in both the stromal and thylakoid et al., et al., but interaction of with precursor proteins to on the A complex on the is of with et al., 1997; and addition of some of the precursor is into the lumen et al., are with precursor proteins that are by and of the of the thylakoid is can be one of the machinery not by the thylakoid machinery is the ability to pathway proteins For to a targeting is by the machinery in the protein is to transport by the thylakoid machinery et al., 1997; for the in role of from of the is a that is in transport of and the and The encodes a protein to and et al., in that transport et al., 1996; et al., The essential in role of also has been with the of a and 1998). This also has a but even in thylakoid than The pathway 1) some with the signal of the and and import into the is into a the transit that in a and and 1991). of the transit complex but is for into the membrane in Cline and Henry, 1996). a of the to be necessary for both the of the transit complex and the of into the and studies that is with in the transit complex et al., of from stromal the ability of stroma to transit complex and et al., to the and to as essential or for targeting to the membrane (Figure 3). In other the is it can and and thus from the which is and and the which also to et al., and are two of stromal et al., of which one is with and may in insertion of membrane The second in a that is in of that the with a protein but et al., from other that and In of the that is in the is that with its A for the of and Proteins via Proteins and and are to across the membrane through a of and This pathway is referred to as the of the which of and stromal and a membrane also are This pathway is referred to as the and require the membrane protein for This pathway is the pathway the is the for A pathway mediates the insertion of the membrane proteins and by a for is in the that are but not to on a and are the stromal of and of the thylakoid pathway are but the of and is (Figure 3). A complex of and is sufficient to the transit complex et al., 1998). are also necessary for although they are not additional stromal is to the et al., 1998). One for the additional be a of the which is with the and is for et al., and additional studies are to the and of the even at this stage, it is that chloroplasts have not in The pathway that the thylakoid as for transport of lumenal proteins 1) is referred to as the pathway (Figure 3). This pathway to be a of its and the that proteins to be by the pathway are from This is in to the thylakoid pathway, which proteins that are also in and Cline and Henry, 1996). of a of the pathway machinery that it is of a to be in the pathway and The a membrane protein a single domain the and acidic domain et al., is in to a in the envelope and and is with its and domain in the a role as a et al., Although is no direct that as a that to specifically transport on the pathway that it is directly in the transport and is the between and proteins of in a of et to that transport are in protein are proteins, or that contain complex 1996). these proteins a in their signal peptides where is essential for their translocation et al., et al., As all pathway also the in their signal it has been that the signal peptide from which contains the directs and transport across thylakoid membranes on the pathway and 1998). work the of a pathway in of in membrane targeting of the and in localization of two proteins the a of as essential of the et al., 1998). of genes two additional and in of a of proteins et al., 1998). The of three in is with the of two in most and the that are at least two proteins in A also is in transport and 1998). The encodes a protein in and to This the that proteins in as of the machinery can be from information the transport The contains a that encodes a protein with membrane-spanning of the of a of proteins et al., 1998). of the protein have not been is to proteins in the of two and The of similarity between thylakoid and both in and to be One between the is no that of the a Although the that proteins are into via a of is the various thylakoid pathways are distinct or have common One is that the pathways at the the membrane machinery through which translocation A in the et al., and in et al., where and pathways or with signal peptide in that the insertion of and insertion of share at least one common and The of that is not but is one is is not by and is of et al., these two pathways at the membrane, the from that in where the pathway a of as as et al., 1998). of as a common for both the and of the signal peptide of which contains a is across the membrane at in a at the and in of et al., 1998). to in that thylakoid pathway transport, do not pathway transport and the that the two transport is that protein machinery hydrophobic domains that insertion and 1996). the and pathways hydrophobic signal peptides for targeting that the initiates transport via a which is a of et al., and and is the for insertion and for similarity in the and a that machinery from a common two have hydrophobic targeting peptides that insertion mechanisms is that to be of a pathway is not a The genetic of in in which genes are can For the and is in transport on the and chloroplasts are of membranes and are also impaired in plastid the of and translocation is to secondary to this may require the of genetic such as the of or in which of specific pathways is proteins are by hydrophobic sequences, for pathway proteins have domains with of signal that N-terminal domains, hydrophobic domains and 1996; et al., and domains for by the processing protease (Chaal et al., 1998). that pathway is by the signal peptide et al., et al., transport is by the sequence of the One between and pathway signal peptides is the in the N-terminal domain of pathway The of one or both by transport on the pathway et al., et al., The is also with the as signal peptides that direct transport on both pathways have been et al., 1997; et al., although to the pathway targeting to the pathway a have been the of in signal The first that the of and a in the domain to the et al., The second that the hydrophobic region of the signal peptide is for et al., the sequence of mature proteins can also For signal pathway proteins are at all et al., et al., In one case, which contains a targeting sequence to the mature not even for transport and This that the mature protein proper by the Targeting through the may on the of the targeting The essential for transit complex in the of et al., with as with a with the of the peptides et al., as has been found for in other systems. to two lumenal proteins that do not require for Interestingly, to which is to these studies be to that for transport, they the first of thylakoid pathway the that are two distinct of et al., the of which with peptide studies to the two The ability of one of to interact in the of protein as to the of is not pathways for the of thylakoid Although it is that such a system, it is that the pathways exist to the specific of the of proteins that For the a of membrane proteins In the to be to the of membrane proteins et al., for the lumenal proteins, the pathway of protein of the has been that proteins, such as are and that the pathway, but not the pathway, can transport proteins et al., studies such a and that a is across the pathway at the of et that the with is by the pathway it is at the of it is not by the pathway to The ability of the pathway to transport proteins to be by the system, in which precursor proteins to with in the their transport across the membrane 1996). The that large domains can be via the pathway has for the of transport translocation of not across the thylakoid membrane and 1998). In most cases, the pathways for plastid protein transport and the have been through in is also that many of the mechanisms be through the are to transport in One and to with in studies is the by are essential for of the as be This can be by et al., 1996). the of genes that of the translocation of isolated plastids can of the import process are The of genetic and in by that the to be the that protein import into et al., studies also are needed to identify of the translocation of mutants in envelope translocation thought to be of the essential of plastid but of et have that it is As genetic have been to thylakoid with have they the general of and but genetic are a into the of protein localization that in For although the of mutants generally are with in are some In the of a pathway are removed or in the precursors do not enter The and although of pathway and pathway and and 1998). deficient in et al., or lacking or et al., are to in proteins are by mechanisms that are of essential in genetic the for understanding mechanisms and pathways that exist in One is that the plant cell to the of a translocation pathway by other that can for the For in the chaperones and the of et al., which the is from and 1991). to is that for for the protein for and One between in and in that the of protein transport mechanisms the between and protein localization For studies of thylakoid protein localization plastids that contain mature thylakoid membranes, in thylakoid proteins are with the of thylakoid has been that from the inner envelope membrane for the of the thylakoid and that of thylakoid membrane even be first in the envelope et al., 1991; for a review of plastid in this this is at in thylakoid precursor proteins stromal such as that are for the compartment. and can be best by a of in and in studies of plastid protein to the mechanisms that are in protein translocation in and the of provided on the in the has been by from the and the at the of and by the and the of
Source: The Plant Cell
DOI: 10.2307/3870884
Authors: Kenneth Keegstra, Kenneth Cline
Institutions: Michigan State University, University of Florida
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2028266323