2.5.1 Protein Sorting and Secretion
A common feature in cells of prokaryotic and eukaryotic origin is the transport of proteins from their site of synthesis, mostly the cytoplasm, to other destinations either inside or outside the cell. This combination of protein sorting, intracellular transport, and secretion to the surrounding environment, is a complex process, which includes numerous steps and requires sophisticated logistics.
In eukaryotic cells, protein synthesis takes place to a large extend on ribosomes that are initially freely mobile in the cytosol. Some proteins are synthesized as immature pre-proteins with an amino-terminal, transient targeting sequence (also termed signal sequence or leader sequence), which serve to direct the protein to its proper destination. The subsequent transport of these proteins to their final destination is then realized using one of two very distinct pathways (Alberts 1994).
The journey on the first pathway begins with the release of the finished protein into the cytosol. There the protein remains, unless it contains a sorting signal for further transport to the mitochondria, peroxisomes, chloroplasts (in plants) or nucleus (Kelly 1985; Alberts 1994).
Because during this thesis, evidence was found, that one of the studied proteins (PIG7p) could be located in the nuclei of the host cells, the general protein transport into the nucleus is further described below.
Approximately 17% of all eukaryotic proteins may be imported into the cell nucleus (Cokol et al. 2000). Transport into the nucleus is thus a common occurrence and has been described and reviewed frequently (Yoneda et al. 1999; Hodel et al. 2001; Macara 2001; Kosugi and Ohashi 2002). To facilitate their transport into the nucleus, these proteins contain a nuclear localization signal (NLS), which is explained in more detail in chapter 2.5.2. Transportation of macromolecules, such as proteins, from the cytoplasm to the nucleus occurs through nuclear pore complexes (NPCs) (Görlich and Kutay 1999). The NPCs create aqueous channels across the nuclear envelope through which macromolecular transport between nucleus and cytoplasm occurs (Davis 1995). Small molecules diffuse passively through the NPCs, while nuclear import and export of larger proteins is an active, signal-mediated process. These larger proteins must bind to receptors at the nuclear pores and be actively transported through the NPCs into the nucleus (Bonner 1978; Lang et al. 1986; Fried and Kutay 2003).
Traveling along the second pathway, (pre-)proteins destined for secretion, the cell organelles, the plasma membrane, or the cell membrane, are first transported to the endoplasmic reticulum (ER). These proteins enter the ER during their synthesis, facilitated by their (mostly N-terminal) leader sequence (Kelly 1985). The proteins are then either retained in the ER or transported to the Golgi apparatus after proper folding and modification (Alberts 1994;
Kostova and Wolf 2003). Upon arrival in the Golgi apparatus, the proteins may be retained anew, sorted to the lysosomes and endosomes or, upon fusion of the secretory vesicles membranes with the plasma membrane, sorted to the extracellular space (Alberts 1994).
The importance and function of targeting sequences such as the N-terminal leader sequences and Nuclear Localization Signals are described in more detail in chapter 2.5.2 below.
2.5.2 Targeting Sequences
For different protein destinations, varying types of targeting sequences are known to exist.
During this project, attention was focused on two specific types of sequences: N-terminal leader sequences and Nuclear Localization Signals (NLS).
The existence of an N-terminal leader sequence was a pre-requisite characteristic of the genes studied during this thesis. The interest in NLS stems from the fact that, such a sequence was found in PIG7p, which in turn could be located in the nucleus of the host plant.
Leader sequences of proteins are small N-terminal sequences (18 to 30 amino acids) containing information about the future localization of the respective protein. In most cases of protein export, the leader sequence is responsible for the passage into or through a membrane (Muesch et al. 1990; Alberts 1994; Izard and Kendall 1994). However, in most cases, the N-terminal leader sequence is not part of the mature protein and only in some cases the leader remains part of the mature protein (Muesch et al. 1990). A protein carrying this leader sequence is called a pre-protein, and is a transient precursor to the mature protein. Leader sequences of proteins designated for secretion are cleaved from the protein by a protease while passing through the ER (Burgess and Kelly 1987; Alberts 1994).
As described above, certain proteins, despite being larger than the passive diffusion limit, can accumulate within the nucleus (Kalderon et al. 1984; Adam et al. 1990; Görlich and Kutay 1999). These observations have led to the discovery of so-called Nuclear Localization Signals (NLS) within the protein, for example in the simian virus 40 (SV40) reviewed by Macara (2001). A NLS is usually a short internal sequence of amino acids located within the protein, which mediates the import of proteins into the nucleus (Cokol et al. 2000). NLS have basic residues and in rare cases hydrophobic residues. It has been shown that the deletion of a NLS disrupts nuclear import (Cokol et al. 2000).
Different types of nuclear localization signals have been described (Macara 2001). They are listed in the following table (Table 2-1). Macara also assumes that many more NLS must exist, but, so far, none have been identified (Macara 2001).
Table 2-1 Nuclear Localization Signal Sequences (Macara 2001)
Signal Proposed consensus sequence Source of reference cited in
Macara (2001) Classical monopartite
NLS
B4,P(B3x),Pxx(B3x),B3(H/P) PSORT II server Classical bipartite
NLS
BBx10(B3x2) PSORT II server
M9 NLS (Y/F/W)x2JxSxZG(P/K)(M/L/V)(K/R) (Bogerd et al. 1999)
Viral NLS RxxRRx1,2RBR (Palmeri and Malim; Truant
and Cullen) Ribosomal L23 NLS VHSHKKKKIRTSPTFRRPKTLRLRRQPKYRRKSAP
RRNK
(Jakel and Gorlich)
B, basic residue (K or R); J hydrophilic residue; Z, hydrophobic residue; x any residue; subscript numbers show number of residues; letters in parentheses can be any order; letters separated by slash are alternate permitted residues.
A NLS may operate using various possible mechanisms. The signal could either bind directly to the nuclear pore channel (NPC) and subsequently be transported through the pores, or the signal could be recognized by soluble receptors, which then transport it through the pores. For permeabilized mammalian cells, it was shown that nuclear protein import requires soluble cytoplasmic factors (Adam et al. 1990; Adam and Adam 1994), of which four different variants are known (see Table 2-2). Most of the proteins that carry other molecules through the NPC are members of the importin β (also termed karyopherin β) family (Macara 2001).
Importin β is a complex carrier able to transport its cargo either via direct bonds (at defined sites) or with the help of an adapter protein (importin α) that binds to the NLS of the cargo.
Table 2-2 Soluble Import Factors (Macara 2001)
Name (Proposed) function Source of reference cited in
Macara (2001) Importin α (karyopherin α,
Kapα, PTAC58)
Adapter protein 55kDa binds to NLS (Adam et al. 1990; Adam and Adam 1994; Görlich et al. 1994;
Moroianu et al. 1995) Importin β, karyopherin β,
p97, PTAC97)
Binds to nucleporins, directs cargo carrier or cargo carrier via adapter protein
(Chi et al. 1995; Görlich et al.
1995; Radu et al. 1995)
Ran (TC4) GTPase (Melchior et al. 1993; Moore and
Blobel 1993) NTF2 (p10, pp15) Part of a multi-component system of
cytosolic factors that assembles itself at the pore complex during nuclear import
(Paschal and Gerace 1995; Paschal et al. 1996)
One example of the importance of NLS in plant pathogen interactions is the avirulence protein, RRS1-R, of Arabidopsis thaliana. RRS1-R, which has a putative NLS, confers broad-spectrum resistance to several strains of the causative agent of bacterial wilt, Ralstonia solanacearum (Deslandes et al. 2003). This bacterium is a devastating plant pathogen with global distribution and a wide host range. A physical interaction between RRS1-R and PopP2, an avirulence protein of R. solanacearum and a type III effector targeting the plant nucleus, has been identified. The co-localization of the Avr protein PopP2 and the RRS1 protein, in the nucleus of Arabidopsis protoplasts, has been shown using GFP transformants (Deslandes et al. 2003).
The above example highlights another aspect of plant pathogen interactions and protein transport. As can be seen in the case of PopP2, mechanisms exist which allow for the export of alien pathogen proteins into the nucleus of the host cells, not just into the cell nucleus of the pathogen itself.
The results of this thesis indicate that one of the studied proteins, PIG7p (RTP1p) has an NLS targeted at the host cell nucleus. This protein can be found at the plant fungal interface as well as in the host nucleus itself, a phenomenon that has never been observed in plant fungal interactions before. Therefore, the process of alien protein export into host cells is described in chapter 2.5.3 below.
2.5.3 Alien Protein Transport into Host Cells
The results of this thesis indicate the presence of a fungal protein from Uromyces fabae in the nucleus of its host plant, Vicia faba. To date, very little is known about the transport of fungal proteins into plant nuclei. Thus it may be helpful to study known examples of alien protein transport into host cells by other pathogens.
For example, alien protein transport into host cells has been described in some detail with regard to the plant pathogenic soil bacterium, Agrobacterium tumefaciens, the gram-negative bacterial tomato pathogen, Xanthomonas campestris (Szurek et al. 2002) and the simian virus, SV40 (Herrera-Estrella et al. 1990; Citovsky et al. 1992; Citovsky et al. 1994; Ballas and Citovsky 1997). Despite the substantial differences between bacteria, viruses and fungi, the findings made in connection with these three pathogens are described below, as analogies with the phenomenon observed in U. fabae during this thesis may exist.
A. tumefaciens is capable of transferring its DNA and some of its proteins (VirD1 and VirD2) into the cell nucleus of the host plant, tobacco. Supporting evidence is provided by experimental findings that the amino-terminal portion of the A. tumefaciens VirD2 protein is able to direct a β-galactosidase fusion protein into the tobacco nucleus (Herrera-Estrella et al.
1990).
In case of gram-negative bacterial (plant) pathogens it is known that sophisticated strategies are employed to invade and colonize plants. Many of these pathogens use a specialized secretion system as a molecular syringe to inject effector proteins directly into the host cell reviewed by Büttner and Bonas (2002). These effector proteins modulate a variety of host cellular pathways, such as rearrangements of the cytoskeleton and defense responses (Vivian and Arnold 2000; Büttner and Bonas 2003). One example is an effector protein from the bacteria, Xanthomonas campestris pv. Vesicatoria, which can be located in the nuclei of infected pepper leaves (Minsavage et al. 1990; Vivian and Arnold 2000; Szurek et al. 2002).
This gene belongs to an avirulence gene family, which encodes proteins targeted to plant cells by the abovementioned specialized bacterial secretion apparatus. A member of this gene family is also found in Xanthomonas oryzae pv. Oryzae, where it is targeted to the host cell nucleus and has the potential to interact with the host DNA (Yang et al. 2000).
Another well-studied pathogen is the simian virus, SV40. In order to infect monkey- and other mammalian cells, SV40 transfers DNA and the T-antigen into its host. In addition, virion particles and viral structural proteins of SV40 can be located in the host nucleus. It is assumed that these viral structural proteins facilitate virion import into the nucleus and viral gene expression (Clever et al. 1993; Yamada and Kasamatsu 1993).
During the infection process, SV40 binds to certain molecules on the cell surface and enters cells via a unique endocytic pathway. SV40 is then delivered to the endoplasmic reticulum (reviewed by (Norkin 1999). Subsequently, SV40 traverses towards the nucleus. Using the cell's own nuclear import machinery, the viral genome enter the nucleus through the nuclear pore complex (NPC) (Yamada and Kasamatsu 1993; Dean 1997; Kasamatsu and Nakanishi 1998). In this context, it has been shown that in order to accumulate in the host cell nucleus, the SV40 large T antigen requires an NLS (Kalderon et al. 1984).
As mentioned previously, in-depth knowledge about alien protein transport into host cell nuclei is limited. Moreover, the function of alien proteins in the plant nucleus can only be speculated upon. This is particularly true when discussing fungal protein export to plant nuclei, although, as will be shown later on in this thesis, fungal pathogen proteins can in fact be found in the host plant nucleus.