Identification of potential regulatory elements for the transport of Emp24p.
To examine the possibility of active recycling of Emp24p between the endoplasmic reticulum (ER) and the Golgi, we sought to identify transport signal(s) in the carboxyl-terminal region of Emp24p. Reporter molecules were constructed by replacing parts of a control invertase-Wbp1p chimera with those of Emp24p, and their transport rates were assessed. The transport of the reporter was found to be accelerated by the presence of the cytoplasmic domain of Emp24p. Mutational analyses revealed that the two carboxyl-terminal residues, leucine and valine (LV), were necessary and sufficient to accelerate the transport. The acceleration was sequence specific, and the terminal valine appeared to be more important. The LV residues accelerated not only the overall transport to the vacuole but also the ER to cis-Golgi transport, suggesting its function in the ER export. Hence the LV residues are a novel anterograde transport signal. The double-phenylalanine residues did not affect the transport by itself but attenuated the effect of the anterograde transport signal. On the other hand, the transmembrane domain significantly slowed down the ER to cis-Golgi transport and effectively counteracted the anterograde transport signal at this step. It may also take part in the retrieval of the protein, because the overall transport to the vacuole was more evidently slowed down. Consistently, the mutation of a conserved glutamine residue in the transmembrane domain further slowed down the transport in a step after arriving at the cis-Golgi. Taken together, the existence of the anterograde transport signal and the elements that regulate its function support the active recycling of Emp24p.
Nakamura
N
N
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.
Yamazaki
S
S
Sato
K
K
Nakano
A
A
Sakaguchi
M
M
Mihara
K
K
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; C093262:EMP24 protein, S cerevisiae; D005656:Fungal Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins