Cell surface adherence and endocytosis of protein transduction domains

Abstract

Protein transduction domains (PTD), such as the HIV TAT and the herpes simplex virus VP22 proteins, are reported to translocate across the membranes of mammalian cells. The mechanism of PTD membrane translocation has largely remained elusive, but recent studies suggest that the reported PTD translocation is due to a fixation artifact. We have constructed and expressed the PTDs VP22, TAT, polyarginine, and polylysine fused to the green fluorescent protein to visualize these proteins in both living and fixed cells. The investigated PTDs strongly adhered to the surface of living cells and were internalized by constitutive endocytosis. No cytosolic or nuclear import of the proteins was detected. In contrast, the PTD-GFP fusion proteins were redistributed to the cytosol and nucleus directly after fixation. Our findings suggest that the PTDs only mediate cell surface adherence, a property shared with many other positively charged macromolecules. The cell surface adherence results in endocytosis and accumulation of proteins in endosomes. We suggest that the biological effects observed for PTD fusion proteins are due to cell surface interactions and internalization of the proteins into cells by classical endocytosis.

Direct evidence for the absence of intercellular trafficking of VP22 fused to GFP or to the herpes simplex virus thymidine kinase

“…49 Similar results have also been recently reported for Tat and other positively charged proteins and peptides. [50][51][52][53] Data presented here on VP22 corroborate these results, but they do not support the view that VP22/GFP can be exported from expressing cells as it has been suggested by Lundberg and Johansson. 49 The flow cytometry experiments performed in our study clearly indicate that the methanol permeabilization allowed VP22/GFP to spread from expressing cells toward neighboring cells (Figure 2).…”

Evaluation of Cell-Penetrating Peptide/Adenovirus Particles for Transduction of CAR-Negative Cells

“…Moreover, several investigations suggested that the apparent energy-independent internalization mechanism of CPPs proposed by the earlier studies were due to artifacts in the cell-fixation (Lundberg, Wikstrom et al 2003;Richard, Melikov et al 2003). Despite the studies conducted to reevaluate the cellular uptake pathway of CPP across the plasma membrane, detailed understanding remains unclear.…”

“…Understanding the internalization mechanism of CPPs is essential for the effective delivery of their cargo. Earlier studies reported that internalization of CPPs occurred in receptor and energy-independent manner (Lundberg, Wikstrom et al 2003;Vives 2003).…”

“…In cases of CPPs coupled to cargo, the internalization mechanism was influenced by the size of the conjugate (Zuhorn, Visser et al 2002). Despite the fact that different CPPs are internalized into cells through endocytosis or energy-independent mechanisms, cellular uptake of these CPPs coupled with high molecular weight cargo occurred via endocytosis (Console et al 2003;Lundberg et al 2003;Takeshima et al 2003). Studies have shown that CPPs coupled to high-molecular-weight maleimidederivatized phospholipid are internalized by endocytosis (Console et al 2003 .…”

“…The mechanism of membrane translocation of CPPs and cargo, however, has yet to be identified definitively (Zorko and Langel 2005;Patel, Zaro et al 2007) and there are conflicting studies regarding whether the CPP internalization mechanism involves energy-dependent endocytosis or a non-endocytic mechanism (Drin, Cottin et al 2003;Richard, Melikov et al 2003;Ziegler, Nervi et al 2005). Despite the fact that different CPPs internalize into cells through different mechanisms (i.e., endocytosis or energy-independent membrane perturbations), recent observations have indicated that internalization of these CPPs occurs via endocytosis when the CPPs are coupled with high molecular weight cargo (Console, Marty et al 2003;Lundberg, Wikstrom et al 2003). These findings suggested that the role of the peptide in this case is may be limited to facilitating attachment of cargo to the cell membrane.…”