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Vol. 11, Issue 5, 1801-1814, May 2000

Sorting to Synaptic-like Microvesicles from Early and Late Endosomes Requires Overlapping but Not Identical Targeting Signals

Anastasiya D. Blagoveshchenskaya, and Daniel F. Cutler*

Medical Research Council Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom

In PC12 neuroendocrine cells, synaptic-like microvesicles (SLMV) are thought to be formed by two pathways. One pathway sorts the proteins to SLMV directly from the plasma membrane (or a specialized domain thereof) in an adaptor protein complex 2-dependent, brefeldin A (BFA)-insensitive manner. Another pathway operates via an endosomal intermediate, involves adaptor protein complex 3, and is BFA sensitive. We have previously shown that when expressed in PC12 cells, HRP-P-selectin chimeras are directed to SLMV mostly via the endosomal, BFA-sensitive route. We have now found that two endosomal intermediates are involved in targeting of HRP-P-selectin chimeras to SLMV. The first intermediate is the early, transferrin-positive, epidermal growth factor-positive endosome, from which exit to SLMV is controlled by the targeting determinants YGVF and KCPL, located within the cytoplasmic domain of P-selectin. The second intermediate is the late, transferrin-negative, epidermal growth factor-positive late endosome, from where HRP-P-selectin chimeras are sorted to SLMV in a YGVF- and DPSP-dependent manner. Both sorting steps, early endosomes to SLMV and late endosomes to SLMV, are affected by BFA. In addition, analysis of double mutants with alanine substitutions of KCPL and YGVF or KCPL and DPSP indicated that chimeras pass sequentially through these intermediates en route both to lysosomes and to SLMV. We conclude that a third site of formation for SLMV, the late endosomes, exists in PC12 cells.

* Corresponding author. E-mail address: d.cutler{at}ucl.ac.uk.

Molecular Biology of the Cell
Vol. 11, 1801-1814, May 2000
Copyright © 2000 by The American Society for Cell Biology


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