Structure-based Functional Analysis Reveals a Role for the SM Protein Sly1p in Retrograde Transport to the Endoplasmic Reticulum

Abstract

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum. INTRODUCTIONMembrane fusion in eukaryotic cells is of fundamental importance for exo-and endocytosis and for maintaining the integrity of intracellular organelles. Essential components of the machinery that mediates lipid bilayer mixing or fusion are members of three protein families that are conserved from unicellular yeast to man: the soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptors (SNAREs), the Sec1/Munc18 (SM) proteins and the Ypt/Rab GTPases. So far, evidence favors the view that SNAREs catalyze membrane fusion (Sutton et al., 1998;Weber et al., 1998;Schuette et al., 2004), whereas Ypt/Rab GTPases and SM proteins regulate upstream processes, termed "tethering," to program the membranes to be fused Pfeffer, 2001). Being first identified in screens for membrane-trafficking mutants in Caenorhabditis elegans (Brenner, 1974), all the SM family members are soluble, peripherally membrane-associated proteins with molecular masses of 60 -90 kDa. There are four SM proteins in the budding yeast and seven in mammalian cells. Although the primary sequence homology within SM proteins is rather low, their three-dimensional structures are highly conserved, suggesting that SM proteins fulfill a general role in membrane fusion (Halachmi and Lev, 1996;Rizo and Sudhof, 2002;Gallwitz and Jahn, 2003).The first evidence regarding SM protein function came from the finding that the mammalian N-Sec1/Munc18 protein binds with high affinity to the SNARE syntaxin1 exclusively in its closed conformation, therefore precluding syntaxin1 from SNARE complex assembly (Hata et al., 1993;Pevsner et al., 1994;Garcia et al., 1994;Dulubova et al., 1999;Yang et al., 2000). In agreement with this, overexpression of N-Sec1/Munc18 i...

Structural basis of Vps33A recruitment to the human HOPS complex by Vps16

“…Similarly, the interaction between yeast Cog4 and Sly1 is required for colocalization of the SNARE proteins that mediate intra-Golgi and Golgi-to-ER retrograde transport: Disrupting this interaction inhibits retrograde trafficking of cargo (49). Although Sly1 was first characterized as modulating anterograde (ER-to-Golgi) vesicle trafficking (50,51), a similar retrograde trafficking defect is observed in sly1-5 mutant yeast bearing a mutation (R452A) in Sly1 domain 3b (52). These observations are consistent with Sly1 domain 3b mediating the interaction with Cog4 in a similar manner to the Vps33A-Vps16 complex.…”

Organization of SNAREs within the Golgi Stack

“…The SM proteins functioning at the Golgi are Sly1 and Vps45. Sly1/Sly1p binds syntaxin 5/Sed5p in the Golgi and syntaxin 18/ Ufe1p in the ER and controls anterograde and retrograde transport between the ER and Golgi and within the Golgi stack Li et al 2005). Vps45 binds syntaxin 16/Tlg2p at the TGN and directs transport from early/ recycling endosomes and late endosomes to the TGN (Simonsen et al 1998;Tang et al 1998;Struthers et al 2009).…”

“…In any case, SM proteins bind cognate SNARE complexes. Sly1p binds the non Q a -SNAREs Use1p/pSLT1p and Sec20p, which are part of the Ufe1p complex (Li et al 2005). It also shows low affinity interactions with the Sed5p t-SNARE components Bos1p and Gos1p and with the v-SNAREs Bet1p and Sft1p, and enhances SNAREpin formation (Dascher et al 1991;Dascher and Balch 1996;Kosodo et al 2002;Peng and Gallwitz 2002;Peng and Gallwitz 2004).…”

Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast

“…This result was expected because Sly1p was shown to interact with the ER SNARE Ufe1p (Yamaguchi et al, 2002) and because the mammalian Sly1 protein copurifies with proteins homologous to Dsl1p and Tip20p . Moreover, Li et al (2005) provided direct evidence for Sly1p being in-volved in Golgi-ER retrograde transport (see pp. 3951-3962 of this issue).…”