The adenovirus E3-6.7K protein adopts diverse membrane topologies following posttranslational translocation.
The E3 region of adenovirus codes for several membrane proteins, most of which are involved in immune evasion and prevention of host cell apoptosis. We explored the topology and targeting mechanisms of E3-6.7K, the most recently described member of this group, by using an in vitro translation system supplemented with microsomes. Here, we present evidence that E3-6.7K, one of the smallest signal-anchor proteins known, translocates across the membrane of the endoplasmic reticulum in a posttranslational, ribosome-independent, yet ATP-dependent manner, reminiscent of the translocation of tail-anchored proteins. Our analysis also demonstrated that E3-6.7K could achieve several distinct topological fates. In addition to the previously postulated type III orientation (N-luminal/C-cytoplasmic, termed NtmE3-6.7K), we detected a tail-anchored form adopting the opposite orientation (N-cytoplasmic/C-luminal, termed CtmE3-6.7K) as well as the possibility of a fully translocated form (N and C termini are both translocated, termed NCE3-6.7K). Due to the translocation of a positively charged domain, both the CtmE3-6.7K and NCE3-6.7K topologies of E3-6.7K constitute exceptions to the "positive inside" rule. The NtmE3-6.7K and NCE3-6.7K are the first examples of posttranslationally translocated proteins in higher eukaryotes that are not tail anchored. Distinct topological forms were also found in transfected cells, as both N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. The demonstration of unexpected topological forms and translocation mechanisms for E3-6.7K defies conventional thinking about membrane protein topogenesis and advises that both the mode of targeting and topology of signal-anchor proteins should be determined experimentally.
Moise
Alexander R
AR
Departments of Medical Genetics, Microbiology and Immunology, and Zoology, Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Grant
Jason R
JR
Lippé
Roger
R
Gabathuler
Reinhard
R
Jefferies
Wilfred A
WA
D000255:Adenosine Triphosphate / Q000378:metabolism; D017503:Adenovirus E3 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D017422:Sequence Analysis, DNA
D017503:Adenovirus E3 Proteins; C087149:E3-6.7K protein, Adenovirus; D008565:Membrane Proteins; D000255:Adenosine Triphosphate