Original Article

Components of a Ubiquitin Ligase Complex Specify Polyubiquitination and Intracellular Trafficking of the General Amino Acid Permease

Correspondence to: Chris A. Kaiser, Department of Biology, 68-533, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139. Tel:(617) 253-9804 Fax:(617) 253-6622 E-mail:ckaiser{at}mit.edu.

Gap1p, the general amino acid permease of Saccharomyces cerevisiae, is regulated by intracellular sorting decisions that occur in either Golgi or endosomal compartments. Depending on nitrogen source, Gap1p is transported to the plasma membrane, where it functions for amino acid uptake, or to the vacuole, where it is degraded. We found that overexpression of Bul1p or Bul2p, two nonessential components of the Rsp5p E3–ubiquitin ligase complex, causes Gap1p to be sorted to the vacuole regardless of nitrogen source. The double mutant bul1 bul2 has the inverse phenotype, causing Gap1p to be delivered to the plasma membrane more efficiently than in wild-type cells. In addition, bul1 bul2 can reverse the effect of lst4, a mutation that normally prevents Gap1p from reaching the plasma membrane. Evaluation of Gap1p ubiquitination revealed a prominent polyubiquitinated species that was greatly diminished in a bul1 bul2 mutant. Both a rsp5-1 mutant and a COOH-terminal truncation of Gap1p behave as bul1 bul2, causing constitutive delivery of Gap1p to the plasma membrane and decreasing Gap1p polyubiquitination. These results indicate that Bul1p and Bul2p, together with Rsp5p, generate a polyubiquitin signal on Gap1p that specifies its intracellular targeting to the vacuole.

Key Words: ubiquitin, Golgi, BUL1, E4, GAP1

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