A dynein mutation attenuates motor neuron degeneration in SOD1(G93A) mice.

Cu/Zn SOD1(G93A) transgenic mice develop phenotypical hallmarks of ALS and serve therefore as an established model to study the molecular mechanisms underlying this disease. Recent reports demonstrate that mutations in the motor protein dynein in Legs at odd angles (Loa) and Cramping (Cra1) mice lead to similar but milder phenotypes. Surprisingly, double transgenic mice (Loa/SOD1(G93A)) have been recently shown to attenuate rather than to accelerate the phenotypical expression of motor neuron degeneration. These results raise the question whether other functional relevant mutations in dynein cause a similar effect. To address this question, we have cross-bred SOD1(G93A) with Cra1/+ mice. These double transgenic mice show an attenuated decline of both motor activity and body weight and an increase of survival time compared to SOD1(G93A) mice. Thus, this study confirms that mechanisms associated with dynein such as retrograde axonal transport may play an important role in SOD1(G93A-) toxicity on motor neurons.

Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life, but is not essential for trans-Golgi network localization.

Proprotein convertases are responsible for the endoproteolytic activation of proproteins in the secretory pathway. The most recently discovered member of this family, lymphoma proprotein convertase (LPC), is a type-I transmembrane protein. Previously, we have demonstrated that its cytoplasmic tail is palmitoylated. In this study, we have identified the two most proximal cysteine residues in the cytoplasmic tail as palmitoylation sites. Substitution of either cysteine residue by alanine interfered with palmitoylation of the other. Palmitoylation of LPC was found to be sensitive to the protein palmitoyltransferase inhibitor tunicamycin but not cerulenin. It was also insensitive to the drugs brefeldin A, monensin and cycloheximide, indicating that the modification occurs in a late exocytic or endocytic compartment. Turnover of palmitoylated LPC is significantly faster (t(1/2) approximately 50 min) than that of the LPC polypeptide backbone (t(1/2) approximately 3 h), suggesting that palmitoylation is reversible. Abrogation of palmitoylation reduced the half-life of the LPC protein, but did not affect steady-state localization of LPC in the trans-Golgi network. Finally, LPC could not be detected in detergent-resistant membrane rafts. Taken together, these results suggest that dynamic palmitoylation of LPC is important for stability, but does not function as a dominant trafficking signal.

Recycling of furin from the plasma membrane. Functional importance of the cytoplasmic tail sorting signals and interaction with the AP-2 adaptor medium chain subunit.

The predominant intracellular localization of the eukaryotic subtilisin-like endoprotease furin is the trans-Golgi network (TGN), but a small fraction is also found on the cell surface. Furin on the cell surface is internalized and delivered to the TGN. The identification of three endocytosis motifs, a tyrosine (YKGL(765)) motif, a leucine-isoleucine (LI(760)) motif, and a phenylalanine (Phe(790)) signal, in the furin cytoplasmic domain suggested that endocytosis of furin occurs via an AP-2/clathrin-dependent pathway. Since little is known about proteins containing multiple sorting components in their cytoplasmic domain, the combination of diverse internalization signals in the furin tail raised the question of their individual role. Here we present data showing that the furin tail interacts with the medium (micro2) subunit of the AP-2 plasma membrane-specific adaptor complex in vitro and that this interaction primarily depends on recognition of the tyrosine-based sorting signal and to less extent on the leucine-isoleucine motif. We further provide evidence that the three endocytosis signals are of different functional importance for furin internalization and retrieval to the TGN in vivo, with the tyrosine-based motif being the major determinant, followed by the phenylalanine signal, whereas the leucine-isoleucine motif is only a minor component. Finally, we report that phosphorylation of the furin tail by casein kinase II is not only important for efficient interaction with micro2 and internalization from the plasma membrane but also determines fast retrieval of the protein from the plasma membrane to the TGN.

Importance of the carboxyl-terminal FTSL motif of membrane cofactor protein for basolateral sorting and endocytosis. Positive and negative modulation by signals inside and outside the cytoplasmic tail.

Membrane cofactor protein (MCP), a widely distributed complement regulatory protein, is expressed on the basolateral surface of polarized epithelial cells, and it is not endocytosed. The carboxyl-terminal tetrapeptide (FTSL) is required for polarized surface expression. The ability of this tetrapeptide to serve as an autonomous sorting signal has been analyzed by adding this sequence motif to the C terminus of an apical membrane protein, the influenza A virus hemagglutinin (HA). The recombinant protein HA-FTSL retained the apical localization of the parental HA protein. Substitution of the complete cytoplasmic tail of MCP for the cytoplasmic tail of HA resulted in the targeting of the chimeric protein (HA/MCP) to the basolateral surface suggesting that the carboxyl-terminal FTSL motif is a weak sorting signal that requires additional targeting information from the membrane-proximal part of the cytoplasmic tail of MCP for redirecting an apical protein to the basolateral membrane domain. In contrast to the native HA, the HA-FTSL protein was subject to endocytosis. The basolateral HA/MCP was also found to be internalized and thus differed from the basolateral MCP. This result suggests that the carboxyl-terminal FTSL motif serves as an internalization signal and that in native MCP sorting information outside the cytoplasmic tail counteracts this endocytosis signal. Substitution of a tyrosine for the phenylalanine dramatically increased the internalization with most of the HA-YTSL protein being present intracellularly. Our results are consistent with the view that the interplay of multiple sorting signals and the modification of a well known targeting signal (YTSL) by amino acid exchange (FTSL) determine the constitutive expression of MCP on the basolateral surface of polarized epithelial cells.

A mono phenylalanine-based motif (F790) and a leucine-dependent motif (LI760) mediate internalization of furin.

The eukaryotic endoprotease furin, a member of the subtilisin-related family of prohormone convertases, is synthesized and transported within the constitutive secretory pathway to the plasma membrane, from where it recycles to the trans-Golgi network (TGN). Previous studies showed that TGN-residence and recycling are mediated by the cytoplasmic tail. Two targeting determinants have been described so far, the acidic signal CPSDSEEDEG783 containing two casein kinase II (CKII) phosphorylation sites and the internalization signal YKGL765. Refined analyses of the cytoplasmic domain of furin, which was mutagenized and tagged to the influenza hemagglutinin and to the membrane cofactor protein (CD46) as reporter molecules reveal two additional internalization determinants, a leucine-isoleucine signal, LI760, and a mono phenylalanine-based motif at F790, which functions without any specific neighboring amino acid sequence. Both signals are capable of independently mediating internalization, as has been shown previously for the tyrosine-based signal. Thus, furin internalization is mediated by at least three independent endocytosis signals.

Sorting of furin at the trans-Golgi network. Interaction of the cytoplasmic tail sorting signals with AP-1 Golgi-specific assembly proteins.

The eukaryotic subtilisin-like endoprotease furin is found predominantly in the trans-Golgi network (TGN) and cycles between this compartment, the cell surface, and the endosomes. There is experimental evidence for endocytosis from the plasma membrane and transport from endosomes to the TGN, but direct exit from the TGN to endosomes via clathrin-coated vesicles has only been discussed but not directly shown so far. Here we present data showing that expression of furin promotes the first step of clathrin-coat assembly at the TGN, the recruitment of the Golgi-specific assembly protein AP-1 on Golgi membranes. Further, we report that furin indeed is present in isolated clathrin-coated vesicles. Packaging into clathrin-coated vesicles requires signal components in the furin cytoplasmic domain which can be recognized by AP-1 assembly proteins. We found that besides depending on the phosphorylation state of a casein kinase II site, interaction of the furin tail with AP-1 and its mu1subunit is mediated by a tyrosine motif and to less extent by a leucine-isoleucine signal, whereas a monophenylalanine motif is only involved in binding to the intact AP-1 complex. This study implies that high affinity interaction of AP-1 or mu1 with the cytoplasmic tail of furin needs a complex interplay of signal components rather than one distinct signal.