In yeast, homologues of the synaptobrevin/VAMP family of v-SNAREs (Snc1 and Snc2) confer the docking and fusion of secretory vesicles at the cell surface. As no v-SNARE has been shown to confer endocytosis, we examined whether yeast lacking the SNC genes, or possessing a temperature-sensitive allele of SNC1 (SNC1 ala43 ), are deficient in the endocytic uptake of components from the cell surface. We found that both SNC and temperature-shifted SNC1 ala43 yeast are deficient in their ability to deliver the soluble dye FM4 -64 to the vacuole. Under conditions in which vesicles accumulate, FM4 -64 stained primarily the cytoplasm as well as fragmented vacuoles. In addition, ␣-factor-stimulated endocytosis of the ␣-factor receptor, Ste2, was fully blocked, as evidenced using a Ste2-green fluorescent protein fusion protein as well as metabolic labeling studies. This suggests a direct role for Snc v-SNAREs in the retrieval of membrane proteins from the cell surface. Moreover, this idea is supported by genetic and physical data that demonstrate functional interactions with t-SNAREs that confer endosomal transport (e.g., Tlg1,2). Notably, Snc1 ala43 was found to be nonfunctional in cells lacking Tlg1 or Tlg2. Thus, we propose that synaptobrevin/VAMP family members are engaged in anterograde and retrograde protein sorting steps between the Golgi and the plasma membrane.

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SummaryThe bacterial tRNA Lys -specific anticodon nuclease is known as a phage T4 exclusion system. In the uninfected host cell anticodon nuclease is kept latent due to the association of its core protein PrrC with the DNA restriction-modification endonuclease EcoprrI. Stp, the T4-encoded peptide inhibitor of EcoprrI activates the latent enzyme. Previous in vitro work indicated that the activation by Stp is sensitive to DNase and requires added nucleotides. Biochemical and mutational data reported here suggest that Stp activates the latent holoenzyme when its EcoprrI component is tethered to a cognate DNA substrate. Moreover, the activation is driven by GTP hydrolysis, possibly mediated by the NTPase domain of PrrC. The data also reveal that Stp can be replaced as the activator of latent anticodon nuclease by certain pyrimidine nucleotides, the most potent of which is dTTP. The activation by dTTP likewise requires an EcoprrI DNA substrate and GTP hydrolysis but involves a different form of the latent holoenzyme/DNA complex. Moreover, whereas Stp relays its activating effect through EcoprrI, dTTP targets PrrC. The activation of the latent enzyme by a normal cell constituent hints that anticodon nuclease plays additional roles, other than warding off phage T4 infection.

Phage T4‐induced anticodon nuclease triggers cleavage‐ligation of the host tRNA(Lys). The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene. Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction‐modification genes (prrA‐hsdM and prrD‐hsdR). Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA. Stp could be substituted by a small, heat‐stable E. coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection. Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC‐encoded core. Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC. Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands. The Hsd‐PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.