Role of Sly1p in the ER-to-Golgi transport step

The SM protein that is involved in the ER-Golgi trafficking pathway in yeast is Sly1p (see Table 1.1). Sly1p was first identified in a genetic screen since a single amino acid substitution (E532K) of a dominant mutant, termed Sly1-20 (Suppressor of Loss of Ypt1 function), was able to bypass the deletion of the Rab protein Ypt1p (Dascher et al., 1991).

This was also the first indication for the functional coupling between SM proteins, SNAREs and Rabs. Sly1p seems to be an essential protein for cell viability since its

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depletion is lethal in yeast (Ossig et al., 1991). In addition, through a targeted siRNA screen, its vertebrate analogue Sly1 has been recently found to be required for constitutive secretion in mammalian cells (Gordon et al., 2010).

Sly1p is thought to be involved in the bi-directional transport between the ER and Golgi (Ossig et al., 1991, Li et al., 2005). Its high affinity binding partners are the syntaxins, Sed5p and Ufe1p, which function in the anterograde and in the retrograde trafficking pathways respectively (see Figure 1.5) (Grabowski and Gallwitz, 1997, Kosodo et al., 1998, Yamaguchi et al., 2002, Bracher and Weissenhorn, 2002). Sly1p has been shown to use solely the mode 2 binding (N-peptide) for its interaction with the monomeric syntaxins.

The crystal structure of Sly1p in complex with the N-peptide of Sed5p has already been determined at a resolution of 3.0 Å (Bracher and Weissenhorn, 2002). This has been the second structure of an SM family protein after the neuronal Munc18a (Misura et al., 2000).

Aside from its closely similar overall fold, Sly1p has sequence insertions compared to Munc18a at residues corresponding to α-helix 20. α-20 together with α-21 have been suggested to act as a lid controlling a function of Sly1p, since these helices partially shield the external surface formed by α-13 and α-14, one of the most conserved surface regions among the Sly1p homologues (Bracher and Weissenhorn, 2002) (Figure 1.6). Interestingly, the single amino acid substitution of the Sly1-20 mutant (E532K) lies on the α-20 as well, suggesting that the lid might be permanently open in the mutant and thereby might bypass a Ypt1p guarded regulatory step (Bracher and Weissenhorn, 2002). Supporting this notion, it has been shown in a later study that the deletion of YPT1 can be suppressed by a Sly1p mutant, in which the entire α-20 is removed (Li et al., 2007). There has been no direct interaction detected between Sly1p and Rabs so far, thus the Rab-related function of Sly1p might be dependent on a physical interaction with a yet undefined effector. In addition, α-20 seems to be highly conserved in fungi, but is absent in other eukaryotes, suggesting that Sly1p might have a lineage-specific functioning in a Rab-dependent tethering step of vesicles (Li et al., 2007).

The crystal structure of the Sly1p/ Sed5p complex demonstrates that Sed5p N-peptide (residues 1-21) interacts predominantly with domain 1 of Sly1p by forming two α-helices separated by residue 5. The helical structure of bound Sed5p is most likely induced by Sly1p since this region is unstructured in solution (Bracher and Weissenhorn, 2002, Yamaguchi et al., 2002). The N-terminal residues of Sed5p (residues 1-10), constituting a

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Figure 1.6: Comparison of Sly1p with Munc18a and the Sly1p homologues

A. Ribbon presentation of Sly1p, superimposed with neuronal Munc18a. Regions superimposing with an r.m.s.d. < 3.5 Å are shown in green, those > 3.5 Å are in grey. Sequence insertions in Sly1p resulting in independent secondary structure elements are in red. The loop region between α-21 and α-22 in Munc18a is shown in yellow for comparison. B. Surface conservation among the Sly1p homologues is plotted to the surface of Sly1p using a scale from green (identical) to white (no conservation). Coils denote the Sed5p N-peptide (yellow) and the helices α-20 and α-21 (red). To point out some conserved residues, the image is also shown re-oriented after a ~150˚ rotation around the vertical axis. Figure is adapted from Bracher and Weissenhorn, 2002.

B

A _I

II

III

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conserved sequence signature among Sed5p homologues (Yamaguchi et al., 2002), bind Sly1p through mainly hydrophobic interactions. The most critical residue for these interactions seems to be Phe10, which is accommodated in a highly conserved hydrophobic pocket by Sly1p. Its mutation into Alanine has been reported to abrogate Sly1p-Sed5p interactions (Yamaguchi et al., 2002), suggesting a nucleation role for Phe10 in adopting the helical binding mode (Bracher and Weissenhorn, 2002).

Several lines of evidence suggest that Sly1p plays a positive regulatory role for the SNARE assembly in the ER-to-Golgi transport. For instance, in vitro SNARE-binding assays have demonstrated that Sly1p prevents Sed5p from forming presumably nonphysiological SNARE complexes (Peng and Gallwitz, 2002). In vivo studies interfering with the interaction between vertebrate Sly1p (Sly1) and Sed5p (Syntaxin 5) orthologues have been shown to induce Golgi fragmentation and/or inhibition of ER-to-Golgi transport (Dulubova et al., 2003, Yamaguchi et al., 2002, Williams et al., 2004). Thus, N-peptide interaction of Sly1p-Sed5p seems to be biologically important and required for the integrity of the secretory pathway. Opposite to this notion, however, a study by Peng &

Gallwitz has suggested that the high-affinity interaction between Sly1p and Sed5p is dispensable and not relevant for proper function in vivo (Peng and Gallwitz, 2004). In this study, various mutant forms of Sly1p and Sed5p, which abolish their N-peptide interaction, have been found fully functional in vivo in the absence of their wild-type counterparts. The strains that have only the mutant copies of either Sly1p or Sed5p have shown no defects in cell growth, the vesicular protein transport or the localization of the SM protein. Since in the same study Sly1p was shown to bind the SNARE motifs of non-syntaxin SNAREs, Bos1p (Qb) and Bet1p (Qc), the interpretation was that Sly1p and/or associated proteins possibly have a ‟bridging” role between the components of the SNARE complex. This might hold true, however, it excludes the possibility that additional regions on Sed5p can be involved in Sly1p binding and can be sufficient for the SM protein to bind Sed5p and act on the SNARE assembly. Possible interpretations of this study will be discussed in the further sections.

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