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 alpha-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.

Figure 1.

Transient Expression of Recombinant ARF1p and ARF1(Q71L)p in Tobacco Protoplasts. (A) Coexpression of the secretory marker α-amylase with either wild-type ARF1p (ARF1-WT) or mutant ARF1(Q71L)p. The top panel shows the secretion index for α-amylase under control conditions (Con) compared with the coexpressed ARF1 proteins. The bottom panel shows the total amount of endogenous ARF1p (Con) compared with the higher protein level when 60 μg of wild-type ARF1 or ARF1(Q71L)-encoding plasmid was electroporated. Note the profound effect of the mutant GTPase on α-amylase secretion in contrast to the wild-type GTPase. (B) Coexpression of the secretory marker with ARF1(Q71L)-encoding plasmid with a dilution series of wild-type ARF1-encoding plasmid. Annotations are as in (A), and the amounts of plasmids are indicated in micrograms below the lanes.

Figure 2.

Manipulation of Secretion and Vacuolar Transport via the Key Molecules Sec12p, Sar1p, and ARF1p. Transient expression in tobacco protoplasts. The secretory marker α-amylase or the vacuolar protein phytepsin was coexpressed with increasing concentrations of Sec12-, Sar1(H74L)-, or ARF1(Q71L)-encoding plasmids. The effector plasmid concentrations are given below each lane in micrograms. Mock-electroporated samples were used as blanks for all α-amylase assays (cells and medium) and are shown for phytepsin gel blots in a separate lane (c). Secretion index values for α-amylase are given in white bars, with standard errors indicated for five independent experiments. For phytepsin, equal volumes of medium (M) and cell (C) samples are compared by protein gel blot analysis. The white arrowhead denotes the unprocessed phytepsin precursor, and the black arrowhead denotes the processed vacuolar form of phytepsin devoid of the N-terminal propeptide (Törmäkangas et al., 2001). Note that all effector molecules cause a dosage-dependent inhibition of α-amylase secretion, whereas secretion of the unprocessed phytepsin precursor to the culture medium is induced specifically by ARF1(Q71L) coexpression.

Figure 3.

Influence of BFA on the Transport of α-Amylase and Phytepsin. Transient expression experiment with tobacco protoplasts incubated with increasing concentrations of the drug BFA. The final concentration is indicated above each lane in micrograms per milliliter. Shown are the α-amylase secretion index (white bars) and total (cells plus medium) α-amylase activity per milliliter of cell suspension (gray bars) compared with phytepsin gel blots of medium (M) and cell (C) samples. Error bars are indicated for five independent experiments. The phytepsin precursor and processed form are annotated as in Figure 2. BFA inhibits α-amylase secretion, phytepsin secretion, and intracellular processing of phytepsin. Note that an induced secretion of phytepsin was not observed.

Figure 4.

Influence of ARF1(T31N) on the Transport of α-Amylase and Phytepsin. Transient expression in tobacco protoplasts. The secretory marker α-amylase or the vacuolar protein phytepsin was coexpressed with increasing concentrations of the plasmid encoding the GDP-restricted mutant of ARF1 (T31N). The plasmid concentration is indicated above each lane in micrograms. Shown are the α-amylase secretion index (white bars) and total (cells plus medium) α-amylase activity per milliliter of cell suspension (gray bars) compared with phytepsin gel blots of medium (M) and cell (C) samples. The phytepsin precursor and processed form are annotated as in Figure 2. ARF1(T31N) inhibits α-amylase secretion, phytepsin secretion, and intracellular processing of phytepsin and is comparable to the effect of BFA. In contrast to the effect of ARF1(Q71L) shown in Figure 2, ARF1(T31N) does not induce phytepsin secretion.

Figure 5.

Propeptides of Sweet Potato Sporamin and Barley Lectin Are Functional at the C Terminus of α-Amylase. (A) Scheme of the six cargo molecules: α-amylase (amy) and its derivatives carrying different C-terminal sorting motifs, including ER retention (amy-HDEL), two vacuolar sorting motifs (amy-spo and amy-bl), and mutated versions of both (amy-spoM and amy-blGG). The sequence-specific vacuolar sorting motif is highlighted by stars, and the two point mutations are indicated with arrowheads. (B) Transient expression experiment showing a comparison of the six cargo molecules. The bottom panel shows the secretion index, and the top panel shows a direct comparison of extracellular (white bars) and intracellular (gray bars) α-amylase activity per milliliter of cell suspension. Note that the intracellular retention of amy-spo is almost complete compared with the leaky retention of amy-HDEL and amy-bl. Error bars are indicated for five independent experiments. Note also that the two mutants amy-spoM and amy-blGG are secreted with approximately similar efficiency as α-amylase.

Figure 6.

Leaky Retention of amy-bl Is Attributable to Saturation of the Sorting Pathway. Transient expression experiment showing a time course for a period of 30 h to compare the transport of amy (top), amy-bl (middle), and amy-spo (bottom) as a function of time. Shown are the intracellular (white squares), extracellular (black squares), and total (gray circles) α-amylase activities per milliliter of suspension. Values are means from three measurements from a single time-course experiment. Note that the secretion of amy-bl starts only after an intracellular threshold level is reached (9 h of expression).

Figure 7.

ARF1(Q71L)-Induced Secretion Occurs Specifically for Sequence-Specific Sorting Signals. Transient expression experiment testing the influence of an increasing concentration of ARF1(Q71L)p on the transport of amy-spo and amy-bl. (A) Extracellular (white bars) and intracellular (gray bars) activities per milliliter of protoplast suspension are shown for amy-spo and amy-bl as a function of increased dosage of ARF1(Q71L)p. The plasmid concentration for the effector is given below each lane in micrograms. Error bars are indicated for five independent experiments. Note an increase in the extracellular level of amy-spo at high concentrations of ARF1(Q71L)p, in contrast to amy-bl, whose secretion diminishes under these conditions. (B) Data from (A) represented as secretion index or total activity per milliliter of protoplast suspension for direct comparison between amy-spo and amy-bl. amy-spo is represented by white bars (right y axis for the secretion index) and amy-bl is represented by gray bars (left y axis for the secretion index). Error bars are indicated for five independent experiments. Note the opposite behavior of the secretion index for the two fusion proteins. Note also that the total amy-bl activity decreases with the increasing dosage of ARF1(Q71L)p, whereas that of amy-spo increases slightly.

Figure 8.

ARF1(Q71L)-Induced Secretion Is Dependent on the NPIRL Motif. (A) Transient expression experiment showing the influence of an increasing concentration of ARF1(Q71L)p on the secretion index of amy-spoM (exhibiting the mutated sorting motif illustrated in Figure 5). The plasmid concentration of the effector is given below each lane in micrograms. The last two lanes represent a positive control carried using the same protoplasts, demonstrating the ability of ARF1(Q71L)p to induce secretion of the wild-type sporamin fusion. Error bars are indicated for five independent experiments. Note that none of the concentrations of ARF1(Q71L)-encoding plasmid leads to induced secretion of amy-spoM (cf. Figure 7B, secretion index for amy-spo). (B) Influence of the drug wortmannin on the transport of cargo molecules in transient expression using tobacco leaf protoplasts. The following cargo molecules were compared under control conditions (white bars) or in the presence of wortmannin (gray bars): amy, amy-HDEL, amy-spo, and amy-spoM. Shown is either the secretion index (top) or the percentage of activity remaining after wortmannin treatment (bottom). Error bars are indicated for five independent experiments. Note the induced secretion of amy-spo and amy-spoM in response to the drug. Note also that the total activity is reduced by the drug in all samples except for amy-spo.

Figure 9.

Vacuolar Sorting of amy-spo. Transient expression experiment to illustrate the intracellular partitioning of amy-spo in control conditions or in the presence of ARF1(Q71L) or the drug BFA. Ten standard transfections were pooled for each condition; a portion was analyzed for the secretion index, and the remainder was analyzed for vacuolar sorting. The secretion index is shown at top. The α-amylase/α-mannosidase ratio is shown as a percentage of the total cell extract (set to 100%) to estimate the degree of amy-spo copurification with the vacuolar marker α-mannosidase. Error bars are indicated for five independent experiments. The protein gel blot (bottom) shows the amount of calnexin detected when equal amounts of α-mannosidase were loaded on SDS-polyacrylamide gels for total cell extracts (T) and purified vacuoles (V).

Figure 10.

Model Illustrating the Effect of ARF1(Q71L)p and Wortmannin on Protein Transport. Scheme illustrating the effect of ARF1(Q71L)p and wortmannin on ER export, Golgi export to the prevacuolar compartment (PVC), and Golgi export to the cell surface (black arrows). Under normal physiological conditions without inhibitors, the majority of vacuolar proteins are exported to the prevacuolar compartment and only a small fraction escapes to the cell surface because of receptor saturation. ARF1(Q71L)p inhibits both vacuolar transport and ER export, resulting in a modest induction of secretion of vacuolar proteins. By contrast, the drug wortmannin inhibits only the vacuolar sorting route and causes more pronounced secretion of vacuolar proteins.