Evidence for Multiple Mechanisms for Membrane Binding and Integration via Carboxyl-Terminal Insertion Sequences

Abstract

Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration.

At the onset of transformation polyomavirus middle-T recruits shc and src to a perinuclear compartment coincident with condensation of endosomes

“…We have previously demonstrated that this fusion protein inserts into microsomal membranes in vitro (Kim et al, 1997) and that a Bcl-2 fusion protein that contains the cytochrome b5 insertion sequence is speci®cally targeted to the ER in Rat ®broblasts, and is functional (Zhu et al, 1996). However, Rat2 ®broblasts expressing the mT/cb5 fusion protein behind an inducible MMTV promoter did not display the early morphologic changes suggestive of transformation that we noted in mT expressing cells (Taylor, Zhu and Andrews, unpublished).…”

“…Thus it is possible that mT mediates some cytoskeletal rearrangements via interactions with these molecules from the perinuclear location. Furthermore, the insertion of mT into the relevant membrane that is necessary for transformation may be dependent upon the interaction of the mT complex with cytoskeletal elements (Andrews et al, 1993;Kim et al, 1997).…”

“…Virus stocks were prepared by harvesting cell culture medium 48 ± 72 h post-infection. A construct expressing mT (or modi®ed to express a mT-cytochrome b5 fusion protein (Kim et al, 1997) from a highly regulated version of MMTV promoter kindly provided by Dr William Muller, McMaster University was transfected into Rat2 cells and stable cell lines were selected in G418 and passaged by standard means.…”

Novel Targeting Signals Mediate the Sorting of Different Isoforms of the Tail-Anchored Membrane Protein Cytochrome b5 to Either Endoplasmic Reticulum or Mitochondria

“…Figure 3A (lanes 1 to 3, þ mbs) shows that Cb5-A and Cb5-D, as well as the control ER TA proteins rat Cb5 (Janiak et al, 1994a;Kim et al, 1997) and rat vesicle-associated membrane protein 2 (Vamp2) (Kim et al, 1999), bound ER membranes in a posttranslational manner, as evidenced by their recovery in bottom fractions of step gradients after centrifugation. By contrast, only a small amount of each of these proteins (O) to (Q) Expressed Cb5-A (O) and endogenous mitochondrial E1b (P) in a portion of a BY-2 cell.…”

“…By contrast, only a small amount (5%) of Cb5-A was recovered with mitochondria. The lack of Cb5-A targeting to mitochondria in vitro was surprising because other Cb5 proteins, including the ERspecific rat Cb5 used as a control ( Figure 3B, lane 3, þ mbs), are known to insert spontaneously into mitochondria and other membrane systems, including liposomes, in vitro (Remacle, 1978;Enoch et al, 1979;Kim et al, 1997). A soluble rat preornithine carbamyl transferase-Protein A fusion (pOCTgPA), which is known to be translocated into the mitochondrial matrix (Janiak et al, 1994a), was efficiently (84%) incorporated into mitochondria, as evidenced by processing of the presequence (cleavage) resulting in the formation of a lower molecular mass polypeptide (lane 3, þ mbs).…”