Function of human surf4 in the early secretory pathway

Abstract

Transport along the early secretory pathway is mediated by vesicles that shuttle proteins and lipids between organelles. Highly organized machineries assure correct trafficking in anterograde and retrograde directions as well as homeostasis of the organelles. A unique position in this system hold transmembrane cargo receptors. They are specialized in recognition of soluble luminal proteins and are able to link them to transport machineries on the cytoplasmic side such as vesicular coats. Cargo receptors are abundant proteins, but their inactivation leads to rather limited secretion phenotypes, illustrating the strict selectivity of receptors for a subset of soluble secretory cargo. Increasing evidence links cargo receptors to human diseases. In humans inactivation of the cargo receptor ERGIC-53 leads to inefficient secretion of the blood coagulation factors V and VIII which is already enough to provoke bleeding disorders. Recently the p24 family member p23 was linked to Alzheimer’s disease by regulating amyloid precursor protein trafficking. These studies show the importance and need to characterize the function of cargo receptors in more detail. The identification of Erv29p in the yeast Saccharomyces cerevisiae as a cargo receptor for pro-α-factor (gpαf) opened new insights into the mechanism of cargo selection by recognizing the Ile-Leu-Val (ILV) sequence motif located in the proregion of gpαf. Furthermore deletion of ERV29 leads to a delay in transport of carboxypeptidase Y (CPY) and proteinase A (PrA) as well as to stabilization of the soluble ER associated degradation (ERAD) substrates CPY* and PrA*. So far Erv29p is the only known cargo receptor required for efficient transport of soluble secretory proteins and efficient degradation of misfolded ERAD substrates, suggesting a much wider function than only packaging correctly folded soluble proteins. The mammalian ortholog Surf4 is poorly characterized and gpαf as the cargo for Erv29p does not allow any speculation about a potential secretory cargo for Surf4 in humans. Therefore characterization of Surf4 would give new enlightenment into the mechansims of protein transport within the early secretory pathway in human cells. In order to characterize Surf4 we localized endogenous Surf4 within the early secretory pathway. Mutational analysis of the conserved di-lysine retrieval motif identified Surf4 to cycle between the ER and Golgi in a lysine signal-dependent manner, similarly to the cargo receptor ERGIC-53. The hallmark of cycling transmembrane proteins is their ability to form homo- and heterooligomers. Well known examples are the hexamerization of ERGIC-53 and heterooligmerization of p24 family members. In search of the function of Surf4 we attempted to identify interacting proteins by Blue Native-PAGE. The ability of Surf4 to form hetrooligomeric complexes with other cycling transmembrane proteins such as members of the p24 family and ERGIC-53, well known to mediate interactions with the machinery required for vesicle formation, opens new insights into the multifunctional behaviour of cargo receptors. Depletion of Surf4 together with ERGIC-53 disrupted the early secretory pathway, as depletion of the p24 family member p25, by redistributing COPI from Golgi and ERGIC membranes. Consequently COPI-mediated retrograde transport is reduced leading to disruption of the Golgi apparatus and reduction in ERGIC structures. To test the cargo receptor function of Surf4 for secretory proteins, pulse-chase analysis was performed with cells depleted of Surf4 by short interference RNA (siRNA). Surf4 depletion resulted in reduced secretion of a subset of secretory proteins, implying cargo-receptor function. Is Surf4 also required for efficient degradation of soluble ERAD substrates as Erv29p? Given that alpha-1-antitrypsin Z variant (A1PiZ) is an ERAD substrate in both yeast and human and is stabilized in Erv29p depleted cells, it is a valid model substrate to study the role of Surf4 dependent stabilization of ERAD substrates in humans. Pulse-chase analysis in combination with Surf4 siRNA-mediated protein knockdown revealed normal degradation of A1PiZ, suggesting no requirement of Surf4 to clear the ER of accumulated soluble ERAD substrates. This work could confirm a potential cargo receptor function for Surf4, while it was not required for efficient degradation of the soluble ERAD substrate A1PiZ. In conclusion the studies on Surf4 revealed that cargo receptors have at least two functions. They assure efficient anterograde transport of secreted proteins by their luminal domain and mediate efficient retrograde transport by controlling COPI recruitment via their cytosolic domain. Thereby cargo receptors facilitate exocytic transport and contribute to organelle maintenace.