Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains - PubMed (original) (raw)

Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains

T Darsow et al. Mol Biol Cell. 2001 Jan.

Free PMC article

Abstract

Transport of proteins through the ALP (alkaline phosphatase) pathway to the vacuole requires the function of the AP-3 adaptor complex and Vps41p. However, unlike other adaptor protein-dependent pathways, the ALP pathway has not been shown to require additional accessory proteins or coat proteins, such as membrane recruitment factors or clathrin. Two independent genetic approaches have been used to identify new mutants that affect transport through the ALP pathway. These screens yielded new mutants in both VPS41 and the four AP-3 subunit genes. Two new VPS41 alleles exhibited phenotypes distinct from null mutants of VPS41, which are defective in vacuolar morphology and protein transport through both the ALP and CPY sorting pathways. The new alleles displayed severe ALP sorting defects, normal vacuolar morphology, and defects in ALP vesicle formation at the Golgi complex. Sequencing analysis of these VPS41 alleles revealed mutations encoding amino acid changes in two distinct domains of Vps41p: a conserved N-terminal domain and a C-terminal clathrin heavy-chain repeat (CHCR) domain. We demonstrate that the N-terminus of Vps41p is required for binding to AP-3, whereas the C-terminal CHCR domain directs homo-oligomerization of Vps41p. These data indicate that a homo-oligomeric form of Vps41p is required for the formation of ALP containing vesicles at the Golgi complex via interactions with AP-3.

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Figures

Figure 1

Screens for ALP pathway components. (A) In wild-type yeast cells, protein trafficking between the Golgi and the vacuole proceeds through two parallel pathways. In the CPY pathway, cargo transits first from the Golgi to an endosomal compartment, where the endosomal t-SNARE, Pep12p, mediates docking and fusion with the endosomal membrane. From the endosome, the CPY pathway continues to the vacuole via a second step in which the vacuolar t-SNARE, Vam3p, mediates docking and fusion with the vacuolar membrane. In contrast, the ALP pathway appears to be a direct Golgi-to-vacuole pathway. Sorting signals in the cytoplasmic domains of the cargo proteins such as ALP and Vam3p are recognized by AP-3 and packaged into vesicles that are then directed from the Golgi to the vacuole without transit through an endosomal compartment. (B) Wild-type cells containing an ALP-Ste13p fusion protein are unable to process alpha factor because the fusion protein is directed to the vacuole through the ALP pathway by virtue of the AP-3 sorting signal of ALP. Disruption of trafficking through the ALP pathway by abrogation of AP-3 function results in retention of the ALP-Ste13p fusion protein in the Golgi and normal alpha factor processing. (C) pep12Δ cells disrupt trafficking through the CPY pathway and are temperature sensitive for growth at 38°C. The additional deletion of AP-3 in pep12Δ cells results in misrouting of ALP pathway cargoes such as Vam3p into the CPY pathway. Vam3p at the endosomal compartment can substitute for Pep12p and rescues the temperature sensitivity of_pep12Δ_ cells.

Figure 2

Vacuolar protein sorting and vacuolar morphology of VPS41 mutants. (A) vps41Δ (WSY41) cells transformed with either complementing VPS41 plasmid (pVPS41), or plasmids containing vps41-18 (pVPS41-18), or vps41-231 (pTD44), were pulse-labeled with [35S]cysteine/methionine and chased for 40 min. CPY and ALP were immunoprecipitated with polyclonal antibodies and analyzed by SDS-PAGE and autoradiography (B). The same strains shown in A were labeled with FM4-64 for 15 min at 30°C and then chased in fresh media for 1 h at 30°C.

Figure 3

VPS41 alleles are defective in ALP intermediate formation. Five hundred OD600 equivalents of_vam3_ tsf cells (TDY27),apl5Δ (GOY8), vps41-18 (WSY41 + pVPS41-18), and vps41-231(WSY41 + pTD44) were spheroplasted, temperature-shifted, lysed, and fractionated (see MATERIALS AND METHODS). Approximately 1 OD600 equivalent of material from each fraction was analyzed by SDS-PAGE and Western blot analysis. The distribution of pALP from each gradient was quantitated using Scion Image 1.62, and the relative amounts of protein contained within the Golgi and vesicle-enriched fractions were calculated. Legend: black bars = vesicle fraction; shaded gray bars = Golgi fraction.

Figure 4

Alignment of Vps41p domains. (A) Vps41p contains two conserved domains, an N-terminal domain that is conserved between all of the Vps41p homologues, designated here as domain I, and a C-terminal clathrin heavy-chain repeat (CHCR) domain, which is found in clathrin heavy-chain , a number of Vps proteins, and in Vps41p and all of its homologues. (B) Domain I alignment with the Vps41p homologues from Homo sapiens (H.s.),Drosophila melanogaster (D.m.), and_Lycopersicon esculentum_ (tomato) (L.e.). The amino acid change found in vps41-231 is indicated with an arrow, and the base change is noted. (C) CHCR alignment with Vps41p homologues. Amino acid changes in both_vps41_ tsf and vps41-18 are blocked in gray and indicated with arrows. In B and C, identity between all sequences is blocked in black, and similarity between a subset of the sequences is blocked in light gray.

Figure 5

Analysis of Vps41p interactions. (A)_GAL4_-BD fusion with a C-terminal fragment of Apl5p (aa 729–932) and a GAL4_-BD fusion with full-length Vps41p tested against the indicated Vps41p fragments fused in frame with the_GAL4_-AD. β-Galactosidase assays were done in duplicate on multiple transformants for each experiment. (B and C) The indicated GST fusion proteins were purified from E. coli on GSH-Sepharose. (A) Triton X-100–solubilized 13,000 ×_g supernatant fractions from yeast cells expressing either Apl5p-HA (PRY1), Vps41p-HA (DKY25), or Vps41-231-HA protein (TDY30) were incubated with the immobilized GST fusion proteins at 4°C and then washed. The bound proteins were eluted with sample buffer. For Western blotting, 20% of the eluate was loaded per lane. One percent of solubilized extract (total) was loaded as a reference. (B and C) Top panels: a Western blot probed with anti-HA antibodies; bottom panels: Coomassie-stained gels of 10% of the eluted sample indicate the relative amounts of the fusion proteins used in each experiment. The identity of each GST-fusion protein is indicated to the right of the bottom panels.

Figure 6

Analysis of Vps41p oligomeric complex. (A) Lysates were made from 100 OD600 equivalents of wild-type yeast expressing an integrated _VPS41_-HA fusion construct (DKY25) and centrifuged at 100,000 × g to remove insoluble proteins and membranes. The supernatant fraction was applied to the S300 sizing column. Proteins in the fractions were analyzed by Western blotting using antibodies against the HA epitope. The yeast Vps41p-HA profile is compared with recombinantly produced GST-Vps41p. (B) GST fused to either full-length Vps41p (pGSTVPS41) or a truncated form of Vps41p containing amino acids 1–714 (pGSTVPS41T) were induced in E. coli (XLIB). Lysates were made from 20 OD600 equivalents of E. coli and centrifuged at 100,000 × g to remove insoluble proteins and membranes. Supernatant fractions for both GST-Vps41p and GST-Vps41p (aa 1–714) truncation were subjected to fractionation on S300 sizing columns. In both A and B, the relative amounts of proteins in each fraction from the columns were compared using Scion Image 1.62 and plotted as a percentage of total protein. Bottom panels: Western blots of Vps41p-HA (A) and GST-Vps41p (B) from the columns. Arrows indicate the approximate size of the peak fractions in both A and B.

Figure 7

Model for Vps41p protein domain function. Vps41p contains two regions that are particularly conserved between the known Vps41p homologues; an N-terminal domain required for binding of Vps41p to the C-terminal coiled coil containing domain of Apl5p, and a C-terminal clathrin heavy-chain repeat (CHCR) domain that mediates oligomerization of Vps41p. The N-terminal domain of Vps41p has homology to WD-40 repeats found in Sec13p and an unknown WD40 containing ORF, YMR049C. The sequence identity between the Vps41p domain and the WD-40 domains of Sec13p and the protein encoded by YMR049C is shown blocked in black.

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Vps41p function in the alkaline phosphatase pathway requires homo-oligomerization and interaction with AP-3 through two distinct domains - PubMed (original) (raw)