Translocation, protein insertion

The Na/K ATPase has been extensively purified and characterized, and consists of a catalytic a subunit of around 95 kDa and a glycoprotein 0 subunit of approximately 45 kDa (Skou, 1992). The functional transporter exists as a dimer with each monomer consisting of an a and /3 subunit. Hiatt aal. (1984) have su ested that the non-catalytic jS subunit may be involved in the cottect insertion of the a subunit into the lipid bilayer and, therefore, it is conceivable that a modification of the 0 subunit structure may be reflected by changes in the catalytic activity of the a subunit. Therefore, in studies involving the manipulation of tissue glutathione levels, alterations of intracellular redox state may have an effect on substrate binding at an extracellular site on this ion-translocating protein. 

Fig. 1. Model for membrane protein insertion into the ER membrane. In stage I, an N-terminal signal peptide (not shown) has already initiated translocation across the membrane.

Chloroplasts in higher plants have three membranes the outer and inner envelope membranes and the thylakoid membrane. Very little is known about membrane protein assembly into the two envelope membranes (Soil and Tien, 1998). The thylakoid has been better studied and in fact appears to use mechanisms very similar to those found in E. coli for membrane protein insertion (Dalbey and Robinson, 1999). Thus, SRP, SecA, SecYEG, YidC, and Tat homologues are all present in the thylakoid membrane or in the stroma (the Tat system was first identified in thylakoids, in fact). In contrast to E. coli, however, there are thylakoid proteins that appear to insert spontaneously into the membrane, insofar as no requirement for any of the known translocation machineries has been detected (Mant et al, 2001). 

Nilsson, I., Witt, S., Kiefer, H., Mingarro, I., and von Heijne, G. (2000). Distant downstream sequence determinants can control N-tail translocation during protein insertion into the endoplasmic reticulum membrane./. Biol. Chem. 275, 6207-6213. 

Other adhesins of E. coli as antigen 43, AIDA-I, TibA and intimin of enter-opathogenic and enterohemorrhagic E. coli are true afimbrial adhesins i.e. they are integral outer membrane proteins. However, also intimin seems to be involved in invasion of host cells [62, 63], Intimin, which is actually a whole family of adhesins, is the only example of an adhesion that uses a protein (Tir translocated intimin receptor) in the host cell membrane, that is a bacterial protein inserted into the host by the bacterial type 3 protein secretion system [64],... 

The mechanism for translocating bacterial proteins across the Inner membrane shares several key features with the translocation of proteins into the ER of eukaryotic cells. First, translocated proteins usually contain an N-termlnal hydrophobic signal sequence, which is cleaved by a signal peptidase. Second, bacterial proteins pass through the Inner membrane In a channel, or translocon, composed of proteins that are structurally similar to the eukaryotic Sec61 complex. Third, bacterial cells express two proteins, Ffh and its receptor (FtsY), that are homologs of the SRP and SRP receptor, respectively. In bacteria, however, these latter proteins appear to function mainly In the insertion of hydrophobic membrane proteins Into the Inner membrane. Indeed, all bacterial proteins that are translocated across the inner membrane do so only after their synthesis In the cytosol is completed but before they are folded Into their final conformation. 

Table 9.1 provides the values of membrane resistance (/ ), capacitance (Cm), and thickness d) of artificial BLMs and natural cell membranes [11,18]. The resistance of artificial membranes is much higher than that of biological membranes. This results from the presence of translocators such as peptides and proteins in the cell membranes. The resistance of artificial membranes can however be reduced to the levels of natural cell membranes when ion translocators are inserted. Specific capacitance (C ) is the primary criterion to distinguish between solventless BLMs and black lipid films. Table 9.1 exhibits that the specific capacitance of the solventless BLMs (about 0.9 /itF cm ) approaches the values measured for natural cell membranes, and is almost twice the magnitude observed for black lipid membranes. These values of specific capacitance can be used to estimate the hydrocarbon thickness, d, of membranes using the equation... 

Jakes, K.S., Kienker, P.K., Slatin, S.L. and Finkelstein, A., Translocation of inserted foreign epitopes by a chaimel-forming protein, Proc Natl Acad Sci USA 95 (1998) 4321-4326. 

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