Protein sorting at the cell surface

The first endocytic sorting event occurs at the cell surface: transmembrane proteins may be actively endocytosed, internalised with the bulk membrane flow, or prevented from internalising to remain at the plasma membrane. Cytoplasmic domain targeting signals in transmembrane proteins allow clustering into coated pits and endocytosis via clathrin- mediated endocytosis.

I have shown that the cytoplasmic domain of the SIV-TM protein has more than one endocytosis signal: a YXX0 signal in the membrane-proximal region (amino acids 721- 724 of the full length tail), and a second signal or signals in the region between amino acids 744 and 813. The signals are additive, so that together they allow faster endocytosis than either of the signals independently. The presence of more than one internalisation signal in

SrV-TM may therefore allow more efficient concentration of the protein into clathrin-coated pits at the plasma membrane.

Internalisation signals are thought act by binding to the AP2 complex of the clathrin-coated pit. Recent evidence suggests that tyrosine-based internalisation signals bind to the p2 chain of AP2 (Ohno et al., 1997; Ohno et al., 1996; Ohno et al., 1995; Stephens et al.,

1997; Zhang and Allison, 1997). Although yeast 2 hybrid analysis failed to demonstrate binding of the membrane-proximal signal of SIV-TM to |li2, this does not rule out the

interaction of SIV-TM with AP2. In fact, recent surface plasmon resonance experiments suggest that a peptide of 17 amino acids from the cytoplasmic domain of SIV-TM,

including the the membrane-proximal tyrosine-based signal, interacts with the AP2 adaptor complex (Honing and Marsh, unpublished results).

The presence of several internalisation signals in SIV-TM may allow binding to non­ overlapping sites on the same AP2 complex or binding of the cytoplasmic domain to more than one AP2 complex, thus increasing the affinity of the SIVmac239-TM for clathrin- coated pits. It is also conceivable that one signal in the SIVmac239-TM cytoplasmic domain binds to AP2, while another binds to another protein with adaptor function such as P arrestin. Further analysis is now needed to identify the endocytosis signal(s) between residues 744 and 813, and to identify the clathrin-coated pit proteins that interact with these signals.

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The analysis of the endocytosis signals in SIV-TM was performed using chimeric proteins consisting of the extracellular and transmembrane domains, and 4 amino acids of the cytoplasmic domain of the CD4 reporter protein fused to the cytoplasmic domain of SIV- TM. The endocytic properties of chimeras with tmncated or point mutated SIV-TM cytoplasmic domains were studied in order to analyse regions or residues important for internalisation. CD4 chimeras were used for this analysis since expression of the Env protein in cells in the absence of other viral proteins is difficult. Expression of Env may require co-expression of the viral Rev protein, in order to stabilise and allow transport of the mRNA encoding Env from the nucleus into the cytoplasm for translation (Churchill et al., 1996; Felber et al., 1989). Previous studies have shown that the cell surface

distribution of Env in SIV-infected cells, or Env expressed in the absence of other viral proteins using the Semliki Forest virus expression system correlates well with the surface expression of CD4/spacer/SIV-TM chimeras (Sauter et al., 1996). SIV-Env, or

CD4/spacer/SrV-TM chimeras with a tyrosine at position 723 of a naturally truncated Env were detected predominantly in intracellular compartments, while mutants with a cysteine at position 723 showed high levels of Env expression on the cell surface (Sauter et al., 1996). The CD4/spacer/SrV-TM constmcts may therefore show similar endocytic properties to those of the SIV-Env protein. However, although many cytoplasmic domain signals have been identified for the internalisation of transmembrane proteins, other determinants may also be important. For example, protein oligomerisation may be important for the

recognition of sorting signals (see section 1.5.2). While CD4 is believed to be a monomer (suggesting that the CD4/spacer/SIV-TM chimeras are also monomers), Env is thought to form trimers. It will therefore be important to analyse the endocytosis of Env in order to confirm the data generated using the chimeras. Experiments to stably express Env, by co­ expression of Rev, in mammalian cells for the study of SIV-Env endocytosis are currently in progress.

In contrast to SIV-TM, CD4 has a dileucine-based endocytosis signal in its cytoplasmic domain. Although increasing evidence suggests that tyrosine-based internalisation signals function by interaction with AP2 complexes of the clathrin-coated pit, little is known about the mechanism by which dileucine-based signals cluster proteins into coated pits. Although my co-immunoprecipitation experiments showed no association of CD4 with AP2 under the conditions used, there is now some evidence that the cytoplasmic domain of CD4 does indeed interact with adaptor complexes. Recent surface plasmon resonance experiments indicate that a peptide corresponding to the cytoplasmic domain of CD4 (residues 398-417) binds to the AP2 complex (Honing and Marsh, unpublished results). Activation of the CD4 endocytosis signal requires phosphorylation, and the CD4 peptide used in these experiments was therefore constmcted with a phosphoserine residue at position equivalent to residue 408 of CD4 (see figure 1.6.1.2; Pitcher and Marsh, 1998). The CD3y chain, also has a dileucine-based internalisation signal which in vivo requires phosphorylation for

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activation. Dietrich et al. (1997) have recently shown that an immobilised peptide

corresponding to the membrane proximal cytoplasmic region of CD3y binds to both to API and AP2 adaptors. The two leucines of the DKQTLL signal were essential for binding, and the aspartic acid residue was also important. However, serine phosphorylation of the peptide in vitro was not required for adaptor binding (Dietrich et al., 1997). This implies that phosphorylation of the serine residue may be important to expose the dileucine signal in the context of the multimeric T cell receptor complex, but is not important for the binding of CD3y to adaptors. CD4, unlike CD3y is not part of a multimeric complex, and it has been proposed that the serine phosphorylation is crucial for the interaction of CD4 with AP2 (Dietrich et al., 1997). Alternatively, serine phosphorylation may cause a

conformational change in the stmcture of the cytoplasmic domains of both CD4 and CD3y, allowing exposure of the dileucine-based signal for adaptor binding. If serine

phosphorylation is required to cause a conformational change in CD3y and CD4 in vivo, it is likely that (as with CD3y) a non-phosphorylated CD4 peptide may bind AP2 in vitro. The analysis of a non-phosphorylated CD4 peptide in the surface plasmon resonance system will therefore give important information about the mechanism of activation of dileucine-based signals by phosphorylation.

In document Sorting of CD4 and the SIV envelope glycoprotein in the endocytic pathway (Page 178-180)