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. 2006 Oct;17(10):4157-66.
doi: 10.1091/mbc.e06-03-0210. Epub 2006 Jul 19.

Chs5/6 complex: a multiprotein complex that interacts with and conveys chitin synthase III from the trans-Golgi network to the cell surface

Siraprapha Sanchatjate  1 , Randy Schekman
Affiliations

Chs5/6 complex: a multiprotein complex that interacts with and conveys chitin synthase III from the trans-Golgi network to the cell surface

Siraprapha Sanchatjate et al. Mol Biol Cell. 2006 Oct.

Abstract

In Saccharomyces cerevisiae, the polysaccharide chitin is deposited at the mother bud junction by an integral membrane enzyme, chitin synthase 3 (Chs3p). The traffic of Chs3p to the cell surface from the trans-Golgi network (TGN) depends on two proteins, Chs5p and Chs6p, which sort selected cargo proteins into secretory vesicles. We have found that Chs5p forms a large higher-order complex of around 1 MDa with Chs6p and three Chs6 paralogs: Bch1p, Bud7p, and Bch2p. The Chs5/6 complex transiently interacts with its cargo, Chs3p, and the presence of Chs3p in the complex is dependent on every subunit. Chs5p and Chs6p have unique and crucial roles in Chs3p transport because either a chs5delta or chs6delta mutant drastically reduces the level of Chs3p bound to the remaining subunits of the complex. Bch1p and Bud7p appear to have a redundant function in Chs3p transport because deletion of both is necessary to displace Chs3p from the complex. The role of Bch2p in Chs3p binding is the least important. Chs5p is essential for structural integrity of the Chs5/6 complex and may act as a scaffold through which the other subunits assemble. Our results suggest a model of protein sorting at the TGN that involves a peripheral, possibly coat, complex that includes multiple related copies of a specificity determining subunit.

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Figures

Figure 1.
Figure 1.
Purification of a Chs5/6 complex. (A) Purification of the Chs5/6 complex using Chs5-TAP reveals four additional copurified polypeptides. Different fractions of TAP purification from a CHS5-TAP strain and an untagged control strain were analyzed on SDS-PAGE and stained with Sypro Red. (B) Analysis of deletion mutants identified those four polypeptides as members of the Chs6-family proteins: Chs6p, Bch2p, Bch1p, and Bud7p.
Figure 2.
Figure 2.
Subunits of the Chs5/6 complex associate together in a higher-order multiprotein complex. (A) TAP purification using Chs6-TAP reveals the presence of Chs5p, Bch1p, Bud7p, and Bch2p in the purified complex. The presence of Bch1p was detected by Sypro Red staining of SDS-PAGE gel. Because of low protein expression levels, the presence of Bud7p and Bch2p was detected using epitope-tagged (HA) versions of Bud7p and Bch2p, whose chromosomal loci were replaced with BUD7-3xHA and BCH2-3xHA, respectively. Nonspecific protein bands recognized by anti-HA antibody are denoted with asterisks. (B) Size determination of the Chs5/6 complex by gel filtration chromatography. TEV eluates from CHS5-TAP and CHS5-TAP bch1Δ bud7Δ were fractionated on Superose 6 gel filtration column and subsequently affinity purified with S-protein agarose. Purified fractions were analyzed by SDS-PAGE and Sypro Red staining to monitor the comigration of all subunits of the Chs5/6 complex. The approximate native molecular weight was determined from a plot of elution volumes of standards versus natural log of their molecular weights. Vo is void volume as determined by Blue Dextran, 2000 kDa; 1, thyroglobulin, 669 kDa; 2, catalase, 232 kDa; 3, BSA, 67 kDa; and 4, cytochrome C, 12.4 kDa. (C) Self-assembly of Chs5p in large structures. Purified Chs5/6 complexes by Chs5-TAP from the TEV eluate of wild type and chs6Δ bch1Δ bud7Δ bch2Δ quadruple mutant were analyzed on 4–8% gradient blue native PAGE. Several large forms of Chs5p self-oligomerization were monitored by immunoblotting with antiserum against Chs5p and denoted with asterisks.
Figure 3.
Figure 3.
The Chs5/6 complex transiently interacts with Chs3p and other proteins. (A) Transient interaction between the Chs5/6 complex and its cargo, Chs3p, as well as other candidate molecules were captured by in vivo cross-linking with DSP. TAP purification using Chs5-TAP was performed with DSP concentrations at 0, 1, 2, and 5 mM. Purified complexes were incubated with DTT to reverse the cross-linking and then analyzed by SDS-PAGE and immunoblotting. (B) Quantification of data in A. The levels of proteins in the purified fractions were normalized with the levels of input loaded on the gel (1/8000 of total input). Concentrations of DSP are indicated as follows: open bars, 0 mM; light gray-shaded bars, 1 mM; medium gray-shaded bars, 2 mM; and black bars, 5 mM.
Figure 4.
Figure 4.
Comparison between levels of copurified proteins from wild-type and chs6Δ backgrounds. TAP purification using Chs5-TAP was performed with 5 mM DSP in vivo cross-linking. Levels of copurified proteins were analyzed by quantitative immunoblotting and presented as a percentage of the protein levels in the loaded input (1/8000 of total input). Copurified proteins from wild-type and chs6Δ backgrounds are indicated by gray-shaded bars and black bars, respectively.
Figure 5.
Figure 5.
Chs5p is essential for Chs3p binding and structural stability of the Chs5/6 complex. (A) Purification of the Chs5/6 complexes from wild-type and chs5Δ backgrounds using Chs6-TAP was performed with 5 mM DSP in vivo cross-linking. The levels of copurified Chs3p were analyzed by quantitative immunoblotting and shown as percentage of the Chs3p level in the loaded input (1/8000 of total input). Copurified Chs3p from wild-type and chs5Δ backgrounds are indicated by gray-shaded bar and black bar, respectively. (B and C) The presence of Chs5p is critical for association of the other subunits in the Chs5/6 complex. The copurified levels of Bch1p from wild-type and chs5Δ backgrounds are shown in B. TAP purification using Chs6-TAP was performed and analyzed by SDS-PAGE and Sypro Red staining. In C, the copurified levels of Bud7-3xHA by Chs6-TAP from wild-type, chs5Δ, and bch1Δ backgrounds and of Bch2-3xHA from wild-type and chs5Δ backgrounds were analyzed by immunoblotting and compared. All purifications started with equivalent cell numbers; the expression levels of Bud7-3xHA and Bch2-3xHA were slightly decreased in chs5Δ, and Bud7-3xHA was up-regulated in bch1Δ. (D) The Chs6-family proteins are not essential for complex association of the other subunits. Purified complex by Chs5-TAP from wild-type and various CHS6-family mutant backgrounds were analyzed by SDS-PAGE and Sypro Red staining.
Figure 6.
Figure 6.
Interaction between the Chs5/6 complex and Chs3p is required for the anterograde transport of Chs3p from the TGN to the cell surface. (A) The transport of Chs3p to the cell surface in wild type and mutants lacking different subunits of the Chs5/6 complex was assessed. Level of chitin synthesis by CSIII activity at the cell wall was monitored by growth on a Calcofluor plate. Mutant backgrounds are abbreviated as follows: 6, chs6Δ; 1, bch1Δ; 7, bud7Δ; and 2, bch2Δ. (B) Subcellular distribution of Chs3p was analyzed by step sucrose gradient. Total cell lysates from wild type, chs6Δ, bch1Δ bud7Δ, and bch1Δ bch2Δ were overlaid on step sucrose gradient, centrifuged at 55,000 rpm for 2.5 h. Fractions were collected manually from the top and analyzed by SDS-PAGE and quantitative immunoblotting. PM marker: Gas1p m, mature form; Gas1p I, immature form (ER); Golgi marker: Tlg1p.
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