THE VDAC1 TWO STEP:  A "HEEL-TOE" MECHANISM FOR MEMBRANE INSERTION 

R. McCauley 
Department of Pharmacology, School of Medicine, Wayne State University, Detroit, MI 48201

Mutation of either (or both) K234 and K236 in yeast VDAC1 (283 amino acids) prevents its insertion into the outer membrane of the mitochondrion both in vitro and in vivo [1, 2].  Recently, we have reinvestigated the influence of these C-terminal lysines in the membrane insertion of VDAC1. In vitro experiments involving depletion of ATP, mutation of both K234 and K236 to glutamine and competition with a peptide that mimics residues 219-242 of VDAC1 were performed.  All of these conditions prevented insertion of VDAC1 into the outer membrane as judged by its protection from digestion with proteinase k.  However, under each of these conditions, VDAC1 was sufficiently membrane inserted to be resistant to extraction with alkaline sodium carbonate (ASC).  These results suggested that VDAC1 underwent a two step insertion process.  One step was independent of ATP, did not employ the two crucial lysines and left VDAC1 partially membrane inserted.  The other was energy dependent, utilized K234 and K236 and resulted in full membrane insertion. 

Two fusion proteins were constructed to determine which portions of the VDAC1 molecule were involved in the energy-independent partial insertion.  One fusion protein, N-165 GFP, consisted of the first 165 amino acids of VDAC1 fused to the N-terminus of the green fluorescent protein (GFP).  N-165 GFP did not bind to isolated mitochondria nor did it insert into a membrane protected or ASC resistant form.  A second fusion (C-256 GFP) consisted of the C-terminal 256 amino acids of VDAC1 fused to the C-terminus of GFP.  C-256 GFP bound to isolated mitochondria and became resistant to extraction by ASC; however, C-256 GFP could not integrate into the membrane sufficiently to achieve protection from proteinase k.  These results were taken to mean that the C-terminal 118 amino acids of VDAC1 were essential for insertion into the outer membrane.  This hypothesis was tested by constructing the deletion mutant, D1-166 VDAC1.  Under the conditions of our in vitro assay, the 117 amino acid protein was bound to mitochondria in a manner that was resistant to ASC extraction and partially protected from proteinase k.  The insertion of D1-166 VDAC1 did not depend on ATP, and a form of D1-166 VDAC1 in which glutamine replaced the two crucial lysines was able to insert as well as wild type. 

These experiments indicate that VDAC1 undergoes a two step membrane insertion.  First the pre-protein is recognized by the outer membrane and partially inserted via residues present in the C-terminus (i.e. “heel”).  This is consistent with experiments performed by others using C-terminal deletion mutants of VDAC1 [3].   The initial insertion does not require either ATP or K234 and K236.  Final insertion of the N-terminal portion of VDAC1 (i.e. “toe”) to form a membrane b-barrel is dependent on both lysines and ATP.

[1]  Smith, M. et al. (1995) J. Biol. Chem. 270:28331-28336
[2]  Angeles, R. et al. (1999) J. Bioenerg. Biomemb. 31:137-142
[3]  Court, D. et al. (1996) FEBS Lett. 390:73-77 

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