The GTPase ARF1p Controls the Sequence-Specific Vacuolar Sorting Route to the Lytic Vacuole

Abstract

We have studied the transport of soluble cargo molecules by inhibiting specific transport steps to and from the Golgi apparatus. Inhibition of export from the Golgi via coexpression of a dominant-negative GTP-restricted ARF1 mutant (Q71L) inhibits the secretion of ␣ -amylase and simultaneously induces the secretion of the vacuolar protein phytepsin to the culture medium. By contrast, specific inhibition of endoplasmic reticulum export via overexpression of Sec12p or coexpression of a GTP-restricted form of Sar1p inhibits the anterograde transport of either cargo molecule in a similar manner. Increased secretion of the vacuolar protein was not observed after incubation with the drug brefeldin A or after coexpression of the GDP-restricted mutant of ARF1 (T31N). Therefore, the differential effect of inducing the secretion of one cargo molecule while inhibiting the secretion of another is dependent on the GTP hydrolysis by ARF1p and is not caused by a general inhibition of Golgi-derived COPI vesicle traffic. Moreover, we demonstrate that GTP-restricted ARF1-stimulated secretion is observed only for cargo molecules that are expected to be sorted in a BP80-dependent manner, exhibiting sequence-specific, context-independent, vacuolar sorting signals. Induced secretion of proteins carrying C-terminal vacuolar sorting signals was not observed. This finding suggests that ARF1p influences the BP80-mediated transport route to the vacuole in addition to transport steps of the default secretory pathway to the cell surface.

Multivesicular Bodies Mature from the Trans-Golgi Network/Early Endosome in Arabidopsis

“…To answer this, we used a dominant-negative VPS2 mutant (VPS2-DN), which was generated by deleting the C-terminal MIT-interacting motif responsible for the interaction with SKD1 (Obita et al, 2007;Hurley and Yang, 2008). Vacuolar transport of the reporter a-amylase-sporamin (amy-spo) was measured as the secretion index (SI) given by the ratio of amy-spo detected in the culture medium and within the cells (Pimpl et al, 2003). Coexpression of VPS2-DN caused dosage-dependentinduced secretion of amy-spo, indicating that vacuolar transport was blocked ( Figure 7A).…”

“…Established plasmids were used encoding for markers/reporters as indicated: mRFP-VSR2 (Miao et al, 2008), YFP-SYP61 , Man1-RFP (Nebenfü hr et al, 1999), GFP-sporamin (daSilva et al, 2005), mRFP-ARA7 and ARA6-mRFP , and a-amylasesporamin (Pimpl et al, 2003). For new recombinant plasmids, all DNA manipulations were performed according to established procedures.…”

“…For new recombinant plasmids, all DNA manipulations were performed according to established procedures. Coding sequences were amplified by PCR from either first-strand cDNA prepared from 3-d-old seedlings (Pimpl et al, 2003) or existing plasmid DNA. Recipient vectors were cut according to the restriction sites of the fragments and dephosphorylated prior to ligation.…”

Overexpression of theArabidopsisSyntaxin PEP12/SYP21 Inhibits Transport from the Prevacuolar Compartment to the Lytic Vacuole in Vivo

“…After 24 h, cells and incubation medium were separated, and the a-amylase activity was measured in both fractions. The ratio between the extracellular and intracellular enzyme activity was calculated and is shown as the secretion index, established to be a linear function of time at steady state (Phillipson et al, 2001;Pimpl et al, 2003). Fulllength and truncated SYP21 were expressed at similar levels as illustrated by the protein gel blot below the graphs.…”

“…This fusion has been extensively tested and described previously as a BP80-dependent vacuolar transport cargo (Pimpl et al, 2003;daSilva et al, 2005). Protoplasts were transformed with a constant amount of plasmid encoding Amyspo, together with increasing concentrations of plasmids encoding either SYP21DTM (Figure 2A, DTM, gray bars) or full-length SYP21 (Figure 2A, FL, white bars).…”